Antibodies and their uses for diagnosis and treatment of cytomegalovirus infection and associated diseases

ABSTRACT

Anti CMV antibodies are provided. Thus an antibody of the present invention comprises an antigen recognition domain capable of binding an MHC molecule being complexed with a cytomegalovirus (CMV) pp65 or pp64 peptide, wherein the antibody does not bind said MHC molecule in an absence of said complexed peptide, and wherein the antibody does not bind said peptide in an absence of said MHC molecule. Also provided are methods of using the antibodies.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/450,476 filed on Jan. 6, 2010, which is a National Phase of PCTPatent Application No. PCT/IL2008/000437 having International filingdate of Mar. 27, 2008, which claims the benefit of priority of U.S.Provisional Patent Application Nos. 60/929,207 filed on Jun. 18, 2007and 60/907,343 filed on Mar. 29, 2007. The contents of the aboveapplications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof diagnosing and treating cytomegalovirus diseases and, moreparticularly, but not exclusively, to antibodies capable of same.

Of all the human herpesviruses described to date, infection withcytomegalovirus (CMV) is considered to be the main cause of morbidityand mortality. Approximately 70% of the world population are carriers ofthe virus. Primary infection with the virus results in a life longpersistence in a latent form and is therefore generally asymptomatic inhealthy adults. However, some individuals, such as immuno-compromisedorgan transplant recipients, or individuals infected with humanimmunodeficiency virus (HIV), are at high risk of developing lifethreatening CMV disease due to CMV reactivation. In addition, CMV hasemerged in recent years as the most important cause of congenitalinfection in the developed world, commonly leading to mental retardationand developmental disability.

Immunity to CMV is complex and involves humoral and cell-mediatedresponses. Studies showed that both natural killer (NK) cells andcytotoxic T-lymphocytes (CTLs) are of primary importance in preventionof recurrence. Many gene products participate in generating the CTLresponse to CMV infection, however, the high level expressionfrequencies of the viral protein pp65 (e.g., Genbank Accession No.M15120; SEQ ID NO:48) suggests pp65 as the main target of theCTL-mediated immune response. Among all pp65 peptides, CMV specific-CTLactivity in HLA-A2 positive individuals was found to be mainly directedto the peptide pp65₄₉₅₋₅₀₃ (NLVPMVATV; SEQ ID NO:3) (Chee M S et al.,1990).

Cytosolic proteins, usually synthesized in the cells, such as CMV viralproteins, enter the class I MHC pathway of antigen presentation. In thefirst step, ubiquitinated cytoplasmic proteins are degraded by theproteasome, a cytoplasmic multiprotein complex which generates a largeportion of peptides destined for display by class I MHC molecules.Peptides are then delivered from the cytoplasm to the endoplasmicreticulum (ER) by the transporter associated with antigen presentation(TAP) molecules. Newly formed class I MHC dimers in the ER associatewith and bind peptides delivered by the TAP. Peptide binding stabilizesclass I MHC molecules and permits their movement out of the ER, throughthe Golgi apparatus, to the cell surface. This pathway ensures that anycell synthesizing viral proteins can be marked for recognition andkilling by CD8+ CTL.

Characterization of class I MHC-peptide presentation is essential forunderstanding the acquired arm of the immune response. The conventionalstrategy for detecting and studying rare populations of antigen(Ag)-specific CD8+ T cells is the application of tetrameric arrays ofclass I peptide-MHC complexes (Altman J D., et al., 1996; Lee P P etal., 1999).

The diagnosis of diseases associated with CMV infection such asretinitis, pneumonia, gastrointestinal disorders, and encephalitis isbased on clinical, histological, virological and DNA tests.

Current methods of treating CMV in immuno-compromised (e.g.,immuno-suppressed) subjects (e.g., HIV patients, bone marrowtransplanted subjects), especially CMV retinitis, include anti viraldrugs such as Foscarnet (FOSCAVIR®), Cidofovir (VISTIDE®) Valganciclovir(VALCYTE®) Ganciclovir implants (VITRASERT®) Fomivirsen (VITRAVENE®).However, the use of these drugs may be associated with serious sideeffects such as kidney damage, neutropenia and hypocalcemia. Onestrategy of directly targeting CMV associated pathologies includes theuse of HLA-A2-restricted CD8(+) CTLs directed against pp65. However,attempts to use CMV-specific CD8+ T cell clones for killing CMV-infectedretinal pigment epithelial cells have failed (Allart S, et al., 2003;Invest Ophthalmol V is Sci. 44: 665-71).

Additional background art includes U.S. patent application Ser. Nos.11/203,137; 11/074,803; 10/510,229; and 11/582,416 to Reiter Y, et al.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an antibody comprising an antigen recognition domaincapable of binding an MHC molecule being complexed with acytomegalovirus (CMV) pp65 or pp64 peptide, wherein the antibody doesnot bind the MHC molecule in an absence of the complexed peptide, andwherein the antibody does not bind the peptide in an absence of the MHCmolecule.

According to an aspect of some embodiments of the present inventionthere is provided an antibody comprising a multivalent form of theantibody of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as an activeingredient the antibody of the antibody of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of detecting a cell expressing acytomegalovirus (CMV) antigen, comprising contacting the cell with theantibody of the present invention under conditions which allowimmunocomplex formation, wherein a presence or a level above apredetermined threshold of the immunocomplex is indicative of CMVexpression in the cell.

According to an aspect of some embodiments of the present inventionthere is provided a method of diagnosing a cytomegalovirus (CMV)infection in a subject in need thereof, comprising contacting a cell ofthe subject with the antibody of the present invention under conditionswhich allow immunocomplex formation, wherein a presence or a level abovea pre-determined threshold of the immunocomplex in the cell isindicative of the CMV infection in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a disease associated withcytomegalovirus (CMV) infection, comprising administering to a subjectin need thereof a therapeutically effective amount of the antibody ofthe present invention, thereby treating the disease associated with CMVinfection.

According to some embodiments of the invention, the cytomegalovirus(CMV) pp65 or pp64 peptide is set forth by SEQ ID NO:3.

According to some embodiments of the invention, the antigen recognitiondomain comprises complementarity determining region (CDR) amino acidsequences as set forth in SEQ ID NOs:24-26 and 30-32.

According to some embodiments of the invention, the antigen recognitiondomain comprises complementarity determining region (CDR) amino acidsequences as set forth in SEQ ID NOs: 36-38 and 42-44.

According to some embodiments of the invention, the antibody beingconjugated to a therapeutic moiety.

According to some embodiments of the invention, the antibody is attachedto a detectable moiety.

According to some embodiments of the invention, the antibody being anantibody fragment.

According to some embodiments of the invention, the multivalent form isan IgG antibody.

According to some embodiments of the invention, the subject has asuppressed or a compromised immune system.

According to some embodiments of the invention, the CMV infection isassociated with a disease selected from the group consisting ofmononucleosis, retinitis, pneumonia, gastrointestinal disorders, andencephalitis.

According to some embodiments of the invention, the cell is a retinacell, lung epithelial cell, a gastrointestinal epithelial cell or abrain cell.

According to some embodiments of the invention, the subject is animmuno-compromised organ transplant recipient.

According to some embodiments of the invention, the subject is infectedwith human immunodeficiency virus (HIV).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein, the terms “comprising” and “including” or grammaticalvariants thereof are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereof.This term encompasses the terms “consisting of” and “consistingessentially of”.

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the biotechnology and medical art.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1 a-d depict the specificity of recombinant Fab Abs to the MHCclass I (HLA-A2)—CMV pp65-derived peptide (NLVPMVATV; SEQ ID NO:3)complex. FIG. 1 a—A histogram depicting an ELISA assay in which phageFab clones were reacted with HLA-A2/pp65 complexes. Fab clones werereacted against a specific MHC class I-peptide complex(HLA-A2/pp65₄₉₅₋₅₀₃, marked as “CMV”) and control non-specific complexescontaining gp100₂₈₀₋₂₈₈ peptide (SEQ ID NO:4; YLEPGPVTA; marked as“280”). FIG. 1 b—An SDS-PAGE analysis depicting the expression andpurification of HLA-A2/pp65 TCR-like Fabs. SDS-PAGE analysis of thepurified Fab proteins was performed after metal affinity chromatography.Note the intense protein bands purified from phage clone F5 or H9 with amolecular weight of 45 kDa. FIGS. 1 c and d—Bar graphs depicting anELISA assay of the binding of soluble purified Fabs to theHLA-A2-peptide complexes. The soluble clones H9 (FIG. 1 c) and F5 (FIG.1 d) were reacted against a specific complex (HLA-A2/pp65; “CMV”) orcontrol non specific complexes containing the following peptides: EBV(280-288; SEQ ID NO:5; GLCTLVAML), gp100 (280-288; SEQ ID NO:4), hTERT(540-548; SEQ ID NO:6; ILAKFLHWL), gp100 (209-217; SEQ ID NO:7;IMDQVPFSV), hTERT (865-873; SEQ ID NO:8; RLVDDFLLV), Gag (77-85; SEQ IDNO:9; SLYNTVATL), Pol (476-484; SEQ ID NO:10; ILEPVHGV), MART (26-35;SEQ ID NO:11; ELAGIGILTV), XAGE (SEQ ID NO:12; GVFPSAPSPV), TARP (29-37;SEQ ID NO:13; FLRNFSLML), TAX (11-19; SEQ ID NO:14; LLFGYPVYV).Specificity towards HLA-A2/pp65 complex can be observed in each of thetwo clones.

FIGS. 2 a-d are flow cytometry analyses depicting the detection ofMHC-peptide complexes on the surface of APCs using the H9 and F5 solubleFabs. JY or RMAS-HHD cell lines were pulsed with various specific andnonspecific peptides. JY cells (FIGS. 2 a and c) or RMAS-HHD cells(FIGS. 2 b and d) loaded with the CMV pp65₄₉₅₋₅₀₃ peptide (SEQ ID NO:3)or control peptides (“280”, “540”), incubated with the H9 (FIG. 2 a, b)or F5 (FIGS. 2 c, d) Fab respectively. Specific staining of the pp65loaded cells, but not the control cells, is shown. The same type ofassay was performed with 10 different control HLA-A2-restricted peptides(data not shown).

FIGS. 3 a-c are flow cytometry analyses depicting the detection ofMHC-peptide complexes on the surface of JY cells using H9Fab in itsmonomeric or tetrameric forms. The JY cell line was pulsed withdifferent peptides. FIG. 3 a—JY cells loaded with pp65₄₉₅₋₅₀₃ peptide(SEQ ID NO:3). Incubations were with H9Fab monomer and PE-labeled antihuman Fab, or with H9Fab tetramer connected to PE labeled streptavidin.FIG. 3 b—JY cells loaded with pp65₄₉₅₋₅₀₃ peptide (SEQ ID NO:3).Incubations were with H9Fab monomer and FITC-labeled anti human Fab, orwith H9 whole IgG Ab and FITC-labeled anti human Fab. FIG. 3 c—JY cellsloaded with gp100₂₈₀₋₂₈₈ 280-288 (SEQ ID NO:4) as a control. Incubationswere with H9Fab monomer and PE-labeled anti human Fab, H9Fab tetramerconnected to PE labeled streptavidin or with H9 IgG Ab and PE-labeledanti human Fab. Note the specific binding of the H9Fab in its monomericor tetrameric form, as well as the whole IgG H9 Ab to JY cells pulsedwith the HLA-A2-CMV peptide (pp65 495-503) but not with JY cells whenpulsed with the control peptide (gp100 280-288). Also note the increasedavidity of the IgG Ab as compared to the monomeric Fab, or the increasedavidity of the tetrameric Fab form as compared to the monomeric Fabform.

FIGS. 4 a-c are graphs depicting the affinity determination of the H9 Abin its monomeric (FIG. 4 a) or IgG (FIG. 4 b) forms, as detected bysurface plasmon resonance (SPR) analysis. Each of the forms was flowedover the relevant wells at three different concentrations (0.05 μM, 0.1μM, 0.2 μM) of biotinylated HLA-A2-pp65 495-503 complexes. As a control,H9 Ab were flowed over wells which were coated with control biotinylatedHLA-A2/pEBV complexes (FIG. 4 c). Note the absence of binding signal ofthe H9 Ab over the HLA-A2/pEBV complex (the concentration of HLA-A2/pEBVcomplex was 0.2 μM) as compared to the HLA-A2/pp65 complex (theconcentration of HLA-A2/pp65 complex was 0.2 μM).

FIGS. 5 a-c are flow cytometry analyses depicting the detection of Fabsensitivity threshold (FIGS. 5 a-b) and of rare cells bearing thespecific peptide-MHC complex in a heterogenous cell population (FIG. 5c). In order to detect Fab sensitivity threshold, JY cells were pulsedwith various concentrations of pp65₄₉₅₋₅₀₃ peptide (0.65 nM, 0.1 μM, 012μM, 0.25 μM, 0.5 μM, 1 μM or 100 μM), and incubated with H9Fab monomer(at a concentration of 10 μg/ml) and PE-labeled anti human Fab (FIG. 5a), or H9 Fab tetramer (at a concentration of 10 μg/ml) connected to PElabeled streptavidin (FIG. 5 b). Note the significantly lowconcentration of the pp65₄₉₅₋₅₀₃ peptide needed to pulse JY cells inorder to obtain a significant binding with the H9 tetramer [e.g., athreshold of 65 nM) of the pp65 495-503 peptide] or the H9 monomer[e.g., a threshold of 0.1 μM of the pp65 495-503 peptide]. Detection ofrare population of cells bearing the specific MHC-peptide complex was bypulsing JY APCs with the pp65 495-503 peptide and mixing them with APDcells (HLA-A2-B cell line) at different ratios (FIG. 5 c) so as toobtain pre-determined concentrations of cells expressing the specificMHC-peptide complex. The mixed population was stained with H9Fab (at aconcentration of 10 μg/ml), and detection sensitivity was monitored byflow cytometry. Note the specific detection of as low as 5% cellsbearing the specific MHC-pp65 495-503 complex.

FIGS. 6 a-m are flow cytometry analyses (FIGS. 6 a-1) and a bar graph(FIG. 6 m) depicting the detection of the specific HLA-A2/pp65 complexby H9 tetramer (FIG. 6 e-h) or H9 IgG Ab (Data not shown), afternaturally occurring active intracellular processing. HLA-A2 positivefibroblasts were infected with the CMV laboratory strain AD169 (FIGS. 6a, e, i). HLA-A2 positive uninfected fibroblasts were used as a control(FIGS. 6 c, g, k) as well as HLA-A2 negative infected fibroblasts (FIGS.6 b, f, j) or HLA-A2 negative uninfected fibroblasts (FIGS. 6 d, h, l).Incubation were with PE labeled BB7.2 (FIGS. 6 a-d), PE labeled H9tetramer (FIGS. 6 e-h) or anti pp65 FITC mAB (FIGS. 6 i-1) followed bythe secondary antibody FITC-labeled anti mouse IgG, 72 hours afterinfection. Note the specific binding of the H9 tetramer to HLA-A2positive cells following infection with CMV (FIG. 6 e) as compared tothe absence of binding to HLA-A2 negative cells (FIG. 60 or touninfected cells (FIGS. 6 g and h), demonstrating the specificHLA-A2-CMV (pp65 495-503) complex—dependent binding of the H9 antibodyto cells ex vivo. In contrast, note the non-CMV-dependent binding of theBB7.2 Ab to HLA-A2 positive cells [same binding efficacy in the presence(FIG. 6 a) or absence (FIG. 6 c) of CMV peptide], and thenon-HLA-A2-dependent binding of the Anti pp65 Ab in CMV-infected cells[same binding efficacy in HLA-A2 positive (FIG. 6 i) or HLA-A2 negative(FIG. 6 j) cells]. FIG. 6 m—A cytotoxicity assay by which H9 IgG Ab isshown to block virus infected cells killing mediated by specific CTLline. Fibroblast cells were radioactively labeled with S³⁵ methioninebefore infection with the CMV virus and 72 hours later the cells wereincubated with the H9 IgG Ab. CTLs were added at a target (fibroblastcells infected with CMV):effector (CTL) ratio of 1:10 and incubated forfive hours. Cells incubated with W6 Ab (an antibody directed againstHLA-A,B,C) were used as positive control, while cells without any Abincubation served as a reference for the maximum killing rate. Theseresults demonstrate the TCR-like specificity of the H9 IgG Ab tospecific CMV-infected cells.

FIGS. 7 a-t are flow cytometry analyses depicting kinetic assays whichfollow the dynamics between the HLA-A2 extracellular presentation, theHLA-A2/pp65 peptide extracellular and intracellular complex presentationand the pp65 expression, in HLA-A2+ (positive) cells infected with theCMV wild-type (WT) AD169 strain. 36 (FIGS. 7 a-e), 72 (FIGS. 7 f-j), 96(FIGS. 7 k-o), and 120 (FIGS. 7 p-t) hours after infection the cellswere harvested and incubated with the BB7.2 PE labeled Ab (FIGS. 7 c, d,h, i, m, n, r, s), anti pp65 Ab (FIGS. 7 e, j, o, t; intracellular) andH9 IgG Ab (FIGS. 7 a, b, f, g, k, l, p, q) antibodies and analyzed byflow cytometry. FITC-labeled anti mouse antibody and Alexafluor⁴⁸⁸-labeled anti human antibody were used as secondary antibodiesfor the anti pp65 mAb and the H9 IgG Ab respectively. Intracellularstaining was feasible by cells permeabilization.

FIGS. 8 a-t are flow cytometry analyses depicting kinetic assays whichfollow the dynamics between the HLA-A2 extracellular presentation, theHLA-A2/pp65 peptide extracellular and intracellular complex presentationand the pp65 expression, in HLA-A2+ (positive) cells infected with theRV798 mutant strain. 36 (FIGS. 8 a-e), 72 (FIGS. 8 f-j), 96 (FIGS. 8k-o), and 120 (FIGS. 8 p-t) hours after infection cells were harvestedand incubated with BB7.2 PE labeled Ab (FIGS. 8 c, d, h, i, m, n, r, s),anti pp65 Ab (FIGS. 8 e, j, o, t; intracellular) and H9 IgG Ab (FIGS. 8a, b, f, g, k, l, p, q) antibodies and analyzed by flow cytometry.FITC-labeled anti mouse antibody and Alexa fluor⁴⁸⁸-labeled anti humanantibody were used as secondary antibodies for the anti pp65 mAb and theH9 IgG Ab respectively. Intracellular staining was feasible by cellspermeabilization.

FIGS. 9 a-y are flow cytometry analyses depicting kinetic assays whichfollow the dynamics between the HLA-A2 extracellular presentation, theHLA-A2/pp65 peptide extracellular and intracellular complex presentationand the pp65 expression, in HLA-A2+ (positive) uninfected cells (FIGS. 9a-t) or in HLA-A2-(negative) cells infected with the AD169 Wild Typestrain of CMV. Staining with the H9 IgG antibody, BB7.2 antibody or theanti pp65 antibodies was effected in the uninfected cells harvested atparallel times [i.e., 36 (FIGS. 9 a-e), 72 (FIGS. 9 f-j), 96 (FIGS. 9k-o), and 120 (FIGS. 9 p-t) hours] to the cells infected with theviruses as described in FIGS. 7 a-t and 8 a-t, hereinabove. InfectedHLA-A2-(negative) cells were harvested and stained with the H9 IgGantibody, BB7.2 antibody or the anti pp65 antibody at 120 hours afterinfection with the AD169 CMV virus. Extracellular staining with the H9IgG antibody is shown in FIGS. 9 a, f, k, p and u. Intracellularstaining with the H9 IgG antibody is shown in FIGS. 9 b, g, l, q and v.Extracellular staining with the BB7.2 antibody is shown in FIGS. 9 c, h,m, r and w. Intracellular staining with the BB7.2 antibody is shown inFIGS. 9 d, i, n, s and x. Staining with the anti pp65 antibody is shownin FIGS. 9 e, j, o, t, and y. FITC-labeled anti mouse antibody and Alexafluor⁴⁸⁸-labeled anti human antibody were used as secondary antibodiesfor the anti pp65 mAb and the H9 IgG Ab respectively. Intracellularstaining was feasible by cells permeabilization.

FIGS. 10 a-d are bar graphs depicting quantization of the number ofHLA-A2/pp65 complexes inside and on the surface of virus infected cells.The level of fluorescence intensity on stained cells was compared withthe fluorescence intensities of calibration beads with known numbers ofPE molecules per bead, thus providing a mean of quantifying PE-stainedcells using a flow cytometer. Incubations were with BB7.2 PE labeled Ab(FIGS. 10 c and d), and H9 Ab (FIGS. 10 a and b). PE-labeled anti kappaantibody was used as a secondary antibody for the H9 IgG Ab. Thecalculated number of HLA-A2/pp65 complexes inside cells (FIG. 10 a) andon the surface (FIG. 10 b) as well as the number of general HLA-A2complexes inside the cells (FIG. 10 c) and on the surface (FIG. 10 d) ineach time scale, is shown for cells infected with AD169 (WT), RV798(mutant), and uninfected cells.

FIGS. 11 a-o are confocal microscopy images of immuno-fluorescenceanalyses depicting direct visualization of HLA-A2/pp65 complexes in CMVinfected fibroblasts. Infected cells were harvested at five time scalespost infection [24 (FIGS. 11 a-c), 48 (FIGS. 11 d-f), 72 (FIGS. 11 g-i),96 (FIGS. 11 j-1) and 120 (FIGS. 11 m-o) hours]. Intracellular doublestaining were with the H9 Ab and Golgi marker. Secondary Ab for the H9Ab was anti human alexa fluor⁴⁸⁸. Secondary antibody for the Golgimarker was anti mouse alexa fluor⁵⁹⁴. Shown are images of H9 Ab alone(FIGS. 11 a, d, g, j, m), Golgi marker alone (FIGS. 11 b, e, h, k, n) ormerged images of H9 and Golgi marker (FIGS. 11 c, f, I, l, o).

FIGS. 12 a-o are confocal microscopy images of immuno-fluorescenceanalyses depicting direct visualization of HLA-A2/pp65 complexes in CMVinfected fibroblasts. Infected cells were harvested at five time scalespost infection [24 (FIGS. 12 a-c), 48 (FIGS. 12 d-f), 72 (FIGS. 12 g-i),96 (FIGS. 12 j-l) and 120 (FIGS. 12 m-o) hours]. Intracellular doublestaining were with the H9 Ab and the ER marker. Secondary Ab for the H9Ab was anti human alexa fluor⁴⁸⁸. Secondary antibody for the ER markerwas anti mouse alexa fluor⁵⁹⁴. Shown are images of H9 Ab alone (FIGS. 12a, d, g, j, m), ER marker alone (FIGS. 12 b, e, h, k, n) or mergedimages of H9 and ER marker (FIGS. 12 c, f, I, l, o).

FIGS. 13 a-j are confocal microscopy images of immuno-fluorescenceanalyses depicting direct visualization of HLA-A2/pp65 complexes of thesurface (extracellular) of CMV infected fibroblasts. The cells wereextracellularly stained with the H9 Ab, and anti human alexa fluor⁴⁸⁸ asa secondary Ab (FIGS. 13 a-e). Noninfected fibroblast cells were used asa control (FIGS. 13 f-h). Verification of the virus infection was withanti pp65 Ab and anti mouse alexa fluor⁵⁹⁴ as a secondary Ab (FIGS. 13i-j).

FIGS. 14 a-d depict the amino acid sequences (FIGS. 14 a and c; SEQ IDNOs:16 and 18) and the nucleic acid sequences (FIGS. 14 b and d; SEQ IDNOs:17 and 19) of the heavy chain (FIGS. 14 a and b) and the light chain(FIGS. 14 c and d) of Fab H9. The CDRs are shown in red; the constantregions are shown in green.

FIGS. 15 a-d depict the amino acid sequences (FIGS. 15 a and c; SEQ IDNOs:20 and 22) and the nucleic acid sequences (FIGS. 15 b and d; SEQ IDNOs:21 and 23) of the heavy chain (FIGS. 15 a and b) and the light chain(FIGS. 15 c and d) of Fab F5. The CDRs are shown in red; the constantregions are shown in green.

FIGS. 16 a-d are flow cytometry (FACS) analyses depicting the detectionof HLA-A2/pp65 complexes on the surface of virus-infected cells takenfrom patients. PBMCs isolated from BMT recipients and healthy donorswere stained extracellular and intracellular with the H9 Ab and thesecondary anti human alexa fluor⁴⁸⁸ Ab. FIG. 16 a-Confirmation of thecells' typing by staining with anti HLA-A2 (BB7.2) Ab. FIG. 16b-Extracellular staining of the BMT recipient cells with the H9 Ab. Nodetection of HLA-A2/pp65 complexes is seen in the infected cells usingthe H9 Ab. FIGS. 16 c-d-Intracellular staining of both BMT recipients(FIG. 16 c) and health donor cells (FIG. 16 d) with the H9 Ab. Asignificant specific staining with the H9 Ab of the permeabilizedinfected cells is seen in the BMT recipients (FIG. 16 c). In contrast,no staining of the H9 Ab is seen in cells of the healthy control.

FIGS. 17 a-i are flow cytometry (FACS) analyses depicting examination ofthe proteasome inhibitor effect on the complexes presentation. Infected(FIGS. 17 a-f) fibroblasts were harvested at three time scales postinfection [48 (FIGS. 17 a, d), 72 (FIGS. 17 b, e), 96 (FIGS. 17 c, f)hours], and treated overnight with 10 μg/ml ALLN(acetyl-leucyl-leucyl-norleucinal) (FIGS. 17 a-c) or remained untreated(FIGS. 17 d-f). The cells were stained with H9 Ab followed by anti humanalexa fluor⁴⁸⁸ as a secondary Ab. FACS analysis shows increasedintensity of the signals after treatment with the proteasome inhibitor(FIGS. 17 a-c), compared to untreated cells (FIGS. 17 d-f). Control,uninfected cells (FIGS. g-i) showed no signal while stained with the H9Ab.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates toantibodies capable of binding MHC molecules being complexed withcytomegalovirus (CMV) pp65 or pp64 peptides which can be used to detectCMV infection and presentation on the cell surface and, moreparticularly, but not exclusively, to methods of diagnosing and treatingdiseases associated with CMV infection.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

While reducing the invention to practice, the present inventors havegenerated human T cell receptor (TCR)-like antibodies directed againstcomplexes of MHC and CMV pp65 or pp64 antigenic peptides which canrecognize cells infected with CMV and thus can be used to diagnose andtreat diseases associated with CMV infection.

As shown in the Examples section which follows, recombinant antibodies[e.g., clones H9 (the amino acid sequence of the heavy chain is setforth by SEQ ID NO:16; the amino acid sequence of the light chain is setforth by SEQ ID NO:18) and F5 (the amino acid sequence of the heavychain is set forth by SEQ ID NO:20; the amino acid sequence of the lightchain is set forth by SEQ ID NO:22] which can specifically recognize MHCmolecules when complexed with CMV pp65-derived peptides such as thepp65₄₉₅₋₅₀₃ (SEQ ID NO:3) were isolated and were found to exhibit finespecificity to soluble or membrane-presented CMV pp65-MHC class Icomplex (Examples 1 and 2 of the Examples section which follows). Inaddition, multivalent forms of these antibodies (e.g., tetrameric Fabsor bivalent IgG) which exhibit increased avidity while preserving thespecificity to the CMV pp65-MHC complex (Example 3 of the Examplessection which follows) were capable of detecting as low as 5% ofsubpopulations of cells bearing CMV pp65 peptide-MHC complexes (Example4 of the Examples section which follows). Cytotoxicity assays usingpp65-specific CD8+ T lymphocytes further demonstrated the specificity ofthe TCR-like antibodies of the invention for CMV pp65-MHC complexes bytheir ability to block killing by the CTLs (Example 6 of the Examplessection which follows). Moreover, the TCR-like antibodies of theinvention enabled one, for the first time, to follow CMV pp65-MHC classI complexes both inside and on the surface of cells infected with CMV(Example 5 of the Examples section which follows). In addition, as shownin FIGS. 7-9 and described in Example 7 of the Examples section whichfollows, the TCR-like antibodies of the invention demonstrated thatthere is no correlation between class I MHC down regulation induced bywild-type virus and the generation/presentation of the virus-specificHLA-A2/pp65₄₉₅₋₅₀₃ complex. Further quantitative data revealed thatspecific HLA-A2/pp65 complexes are being generated in large amounts andaccumulated inside the infected cell in a mechanism that is independentto the overall down regulation of HLA-A2 molecules in these cells(Example 8 of the Examples section which follows). In addition, confocalmicroscopy analysis demonstrated that immediately after CMV infectionspecific HLA-A2/pp65 complexes are being generated and accumulated inthe Golgi compartment and only about 72 hours after infection are theHLA-A2/pp65 complexes displayed on the cell surface (Example 9 of theExamples section which follows). Moreover, as shown in FIGS. 16 a-d anddescribed in Example 12 of the Examples section which follows, theantibodies of the invention were shown to be capable of detectingHLA-A2/pp65 complexes in blood cells of subjects with CMV reactivationdue to immune suppression (e.g., bone marrow transplanted subjects). Inaddition, as shown in FIGS. 17 a-j and described in Example 13 of theExamples section which follows, incubation of cells with a proteasomeinhibitor resulted in increased presentation of the MHC/pp65 complexeson the cell surface.

Thus, according to an aspect of some embodiments of the presentinvention there is provided an antibody comprising an antigenrecognition domain capable of binding a Major histocompatibility complex(MHC) molecule being complexed with a cytomegalovirus (CMV) pp65 or pp64peptide, wherein the antibody does not bind the MHC molecule in anabsence of the complexed peptide, and wherein the antibody does not bindthe peptide in an absence of the MHC molecule.

As used herein, the phrase “major histocompatibility complex (MHC)”refers to a complex of antigens encoded by a group of linked loci, whichare collectively termed H-2 in the mouse and HLA in humans. The twoprincipal classes of the MHC antigens, class I and class II, eachcomprise a set of cell surface glycoproteins which play a role indetermining tissue type and transplant compatibility. In transplantationreactions, cytotoxic T-cells (CTLs) respond mainly against foreign classI glycoproteins, while helper T-cells respond mainly against foreignclass II glycoproteins.

Major histocompatibility complex (MHC) class I molecules are expressedon the surface of nearly all cells. These molecules function inpresenting peptides which are mainly derived from endogenouslysynthesized proteins to CD8+ T cells via an interaction with the αβT-cell receptor. The class I MHC molecule is a heterodimer composed of a46-kDa heavy chain which is non-covalently associated with the 12-kDalight chain β-2 microglobulin. In humans, there are several MHChaplotypes, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24,HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, theirsequences can be found at the kabbat data base, athtexttransferprotocol://immuno.bme.nwu.edu. Further informationconcerning MHC haplotypes can be found in Paul, B. FundamentalImmunology Lippincott-Rven Press.

Cytomegalovirus (CMV) belongs to the human herpesviruses. There areseveral known strains of CMV, including strains 1042, 119, 2387. 4654,5035, 5040, 5160, 5508, AD169, Eisenhardt, Merlin, P T, Toledo andTowne. During viral infection, the expressed viral proteins, e.g., pp65of the CMV AD169 strain [GenBank Accession No. M15120 for nucleic acidcoding sequence (SEQ ID NO:48) and GenBank Accession No. AAA45996.1 foramino acids (SEQ ID NO:50); or GenBank Accession No. P06725 (SEQ IDNO:53)] pp64 of the CMV Towne strain [GenBank Accession No. M67443 fornucleic acid coding sequence (SEQ ID NO:49) and GenBank Accession No.AAA45994.1 for amino acids (SEQ ID NO:51); or GenBank Accession No.P18139 (SEQ ID NO:52)] are subject to proteasomal degradation and theMHC-restricted peptides bind to the MHC molecules [e.g., MHC class I orMHC class II] and are further presented therewith on the cell surface.The pp65 (561 amino acids in length) and pp64 (551 amino acids inlength) proteins of the CMV AD169 and Towne strains, respectively, are99% identical proteins and share the same amino acid sequence fromposition 3-551 of pp64 and 13-561 of pp65.

As used herein the term “peptide” refers to native peptides (eitherproteolysis products or synthetically synthesized peptides) and furtherto peptidomimetics, such as peptoids and semipeptoids which are peptideanalogs, which may have, for example, modifications rendering thepeptides more stable while in a body, or more immunogenic. Suchmodifications include, but are not limited to, cyclization, N terminusmodification, C terminus modification, peptide bond modification,including, but not limited to, CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O,CH₂—CH₂, S═C—NH, CH═CH or CF═CH, backbone modification and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified in Quantitative Drug Design, C. A.Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which isincorporated by reference as if fully set forth herein. Further detailsin this respect are provided hereinunder.

As used herein in the specification and in the claims section below theterm “amino acid” is understood to include the 20 naturally occurringamino acids; those amino acids often modified post-translationally invivo, including for example hydroxyproline, phosphoserine andphosphothreonine; and other unusual amino acids including, but notlimited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,nor-leucine and ornithine. Furthermore, the term “amino acid” includesboth D- and L-amino acids. Further elaboration of the possible aminoacids usable according to the invention and examples of non-naturalamino acids useful in MHC-1HLA-A2 recognizable peptide antigens aregiven herein under.

Based on accumulated experimental data, it is nowadays possible topredict which of the peptides of a protein will bind to MHC, class I.The HLA-A2 MHC class I has been so far characterized better than otherHLA haplotypes, yet predictive and/or sporadic data is available for allother haplotypes.

With respect to HLA-A2 binding peptides, assume the following positions(P1-P9) in a 9-mer peptide:

P1-P2-P3-P4-P5-P6-P7-P8-P9

The P2 and P2 positions include the anchor residues which are the mainresidues participating in binding to MHC molecules. Amino acid residesengaging positions P2 and P9 are hydrophilic aliphatic non-chargednatural amino (examples being Ala, Val, Leu, Ile, Gln, Thr, Ser, Cys,preferably Val and Leu) or of a non-natural hydrophilic aliphaticnon-charged amino acid [examples being norleucine (Nle), norvaline(Nva), α-aminobutyric acid]. Positions P1 and P3 are also known toinclude amino acid residues which participate or assist in binding toMHC molecules, however, these positions can include any amino acids,natural or non-natural. The other positions are engaged by amino acidresidues which typically do not participate in binding, rather theseamino acids are presented to the immune cells. Further details relatingto the binding of peptides to MHC molecules can be found in Parker, K.C., Bednarek, M. A., Coligan, J. E., Scheme for ranking potential HLA-A2binding peptides based on independent binding of individual peptideside-chains. J Immunol. 152, 163-175, 1994, see Table V, in particular.Hence, scoring of HLA-A2.1 binding peptides can be performed using theHLA Peptide Binding Predictions software approachable through aworldwide web interface at hypertexttransferprotocol://worldwideweb(dot) bimas (dot) dcrt (dot) nih (dot) gov/molbio/hla_bind/index. Thissoftware is based on accumulated data and scores every possible peptidein an analyzed protein for possible binding to MHC HLA-A2.1 according tothe contribution of every amino acid in the peptide. Theoretical bindingscores represent calculated half-life of the HLA-A2.1-peptide complex.

Hydrophilic aliphatic natural amino acids at P2 and P9 can besubstituted by synthetic amino acids, preferably Nleu, Nval and/orα-aminobutyric acid. P9 can be also substituted by aliphatic amino acidsof the general formula —HN(CH₂)_(n)COOH, wherein n=3-5, as well as bybranched derivatives thereof, such as, but not limited to,

wherein R is, for example, methyl, ethyl or propyl, located at any oneor more of the n carbons.

The amino terminal residue (position P1) can be substituted bypositively charged aliphatic carboxylic acids, such as, but not limitedto, H₂N(CH₂)_(n)COOH, wherein n=2-4 and H₂N—C(NH)—NH(CH₂)_(n)COOH,wherein n=2-3, as well as by hydroxy Lysine, N-methyl Lysine orornithine (Orn). Additionally, the amino terminal residue can besubstituted by enlarged aromatic residues, such as, but not limited to,H₂N—(C₆H₆)—CH₂—COOH, p-aminophenyl alanine,H₂N—F(NH)—NH—(C₆H₆)—CH₂—COOH, p-guanidinophenyl alanine orpyridinoalanine (Pal). These latter residues may form hydrogen bondingwith the OH⁻ moieties of the CMV pp65 residues at the MHC-1 N-terminalbinding pocket, as well as to create, at the same time aromatic-aromaticinteractions.

Derivatization of amino acid residues at positions P4-P8, should theseresidues have a side-chain, such as, OH, SH or NH₂, like Ser, Tyr, Lys,Cys or Orn, can be by alkyl, aryl, alkanoyl or aroyl. In addition, OHgroups at these positions may also be derivatized by phosphorylationand/or glycosylation. These derivatizations have been shown in somecases to enhance the binding to the T cell receptor.

Longer derivatives in which the second anchor amino acid is at positionP10 may include at P9 most L amino acids. In some cases shorterderivatives are also applicable, in which the C terminal acid serves asthe second anchor residue.

Cyclic amino acid derivatives can engage position P4-P8, preferablypositions P6 and P7. Cyclization can be obtained through amide bondformation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric(Dab) acid, di-aminopropionic (Dap) acid at various positions in thechain (—CO—NH or —NH—CO bonds). Backbone to backbone cyclization canalso be obtained through incorporation of modified amino acids of theformulas H—N((CH₂)_(n)—COOH)—C(R)H—COOH orH—N((CH₂)_(n)—COOH)—C(R)H—NH₂, wherein n=1-4, and further wherein R isany natural or non-natural side chain of an amino acid.

Cyclization via formation of S—S bonds through incorporation of two Cysresidues is also possible. Additional side-chain to side chaincyclization can be obtained via formation of an interaction bond of theformula —(—CH₂—)_(n)—S—CH₂—C—, wherein n=1 or 2, which is possible, forexample, through incorporation of Cys or homoCys and reaction of itsfree SH group with, e.g., bromoacetylated Lys, Orn, Dab or Dap.

Peptide bonds (—CO—NH—) within the peptide may be substituted byN-methylated bonds (—N(CH₃)—CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—),ketomethylen bonds (—CO—CH₂—), α-aza bonds (—NH—N(R)—CO—), wherein R isany alkyl, e.g., methyl, carba bonds (—CH₂—NH—), hydroxyethylene bonds(—CH(OH)—CH₂—), thioamide bonds (—CS—NH—), olefinic double bonds(—CH═CH—), retro amide bonds (—NH—CO—), peptide derivatives(—N(R)—CH₂—CO—), wherein R is the “normal” side chain, naturallypresented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time. According to someembodiments of the invention, but not in all cases necessary, thesemodifications should exclude anchor amino acids.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

Various pp65 or pp64 MHC restricted peptides can be used to form theMHC-CMV pp65 peptide complex. See for example, the peptides described inExamples 10 and 11 of the Examples section which follows (Tables 5-137).

According to some embodiments of the invention, the antibodies recognizea complex formed between the MHC class I molecule (HLA-A2) and the CMVpp65 peptide set forth by SEQ ID NO:3.

The term “antibody” as used herein includes intact molecules as well asfunctional fragments thereof, such as Fab, F(ab′)₂, Fv and scFv that arecapable of specific binding to a human major histocompatibility complex(MHC) class I-restricted CMV pp65 or pp64 epitope. These functionalantibody fragments are defined as follows: (i) Fab, the fragment whichcontains a monovalent antigen-binding fragment of an antibody molecule,can be produced by digestion of whole antibody with the enzyme papain toyield an intact light chain and a portion of one heavy chain; (ii) Fab′,the fragment of an antibody molecule that can be obtained by treatingwhole antibody with pepsin, followed by reduction, to yield an intactlight chain and a portion of the heavy chain; two Fab′ fragments areobtained per antibody molecule; (iii) F(ab′)₂, the fragment of theantibody that can be obtained by treating whole antibody with the enzymepepsin without subsequent reduction; F(ab′)₂ is a dimer of two Fab′fragments held together by two disulfide bonds; (iv) Fv, defined as agenetically engineered fragment containing the variable region of thelight chain and the variable region of the heavy chain expressed as twochains; and (v) scFv or “single chain antibody” (“SCA”), a geneticallyengineered molecule containing the variable region of the light chainand the variable region of the heavy chain, linked by a suitablepolypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art (See for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988, incorporated herein by reference) and arefurther described herein below.

An exemplary method for generating antibodies capable of specificallybinding a CMV pp65 peptide restricted to an MHC-I complex is describedin the Examples section herein below.

In addition, such antibodies may be generated by (i) immunizing agenetically engineered non-human mammal having cells expressing thehuman major histocompatibility complex (MHC) class I, with a solubleform of an MHC class I molecule being complexed with the HLA-restrictedepitope; (ii) isolating mRNA molecules from antibody producing cells,such as splenocytes, of the non-human mammal; (iii) producing a phagedisplay library displaying protein molecules encoded by the mRNAmolecules; and (iv) isolating at least one phage clone from the phagedisplay library, the at least one phage displaying the antibodyspecifically bindable (with an affinity below 200 nanomolar, e.g., below100 nanomolar, e.g., below 50 nanomolar, e.g., below 30 nanomolar, e.g.,below 20 nanomolar, e.g., below 10 nanomolar) to the human majorhistocompatibility complex (MHC) class I being complexed with theHLA-restricted epitope. The genetic material of the phage isolate isthen used to prepare a single chain antibody or other forms ofantibodies as is further described herein below. For example, thegenetic material of the phage isolate can be used to prepare a singlechain antibody which is conjugated to an identifiable or a therapeuticmoiety. According to some embodiments of the invention, the non-humanmammal is devoid of self MHC class I molecules. According to someembodiments of the invention, the soluble form of the MHC class Imolecule is a single chain MHC class I polypeptide including afunctional human β-2 microglobulin amino acid sequence directly orindirectly covalently linked to a functional human MHC class I heavychain amino acid sequence.

Recombinant MHC class I and class II complexes which are soluble andwhich can be produced in large quantities are described in, for example,Denkberg, G. et al. 2002, and further in U.S. patent application Ser.No. 09/534,966 and PCT/IL01/00260 (published as WO 01/72768), all ofwhich are incorporated herein by reference. Soluble MHC class Imolecules are available or can be produced for any of the MHChaplotypes, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24,HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8,following, for example the teachings of PCT/IL01/00260, as theirsequences are known and can be found at the kabbat data basehypertexttransferprotocol://immuno (dot) bme (dot) nwu (dot) edu/, thecontents of the site is incorporated herein by reference. Such solubleMHC class I molecules can be loaded with suitable HLA-restrictedepitopes and used for vaccination of non-human mammal having cellsexpressing the human major histocompatibility complex (MHC) class I asis further detailed hereinbelow.

Non-human mammal having cells expressing a human majorhistocompatibility complex (MHC) class I and devoid of self majorhistocompatibility complex (MHC) class I can be produced using (i) thesequence information provided in the kabbat data base, athypertexttransferprotocol://immuno (dot) bme (dot) nwu (dot) edu/, whichis incorporated herein by reference and pertaining to human MHChaplotypes, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24,HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, (ii)conventional constructs preparation techniques, as described in, forexample, “Molecular Cloning: A laboratory Manual” Sambrook et al.,(1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel,R. M., ed. (1994); Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “APractical Guide to Molecular Cloning”, John Wiley & Sons, New York(1988); Watson et al., “Recombinant DNA”, Scientific American Books, NewYork; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”,Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); and(iii) conventional gene knock-in/knock-out techniques as set forth, forexample, in U.S. Pat. Nos. 5,487,992, 5,464,764, 5,387,742, 5,360,735,5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384,5,175,383, 4,736,866; in International Publications WO 94/23049,WO93/14200, WO 94/06908 and WO 94/28123; as well as in Burke and Olson,Methods in Enzymology, 194:251-270, 1991; Capecchi, Science244:1288-1292, 1989; Davies et al., Nucleic Acids Research, 20 (11)2693-2698, 1992; Dickinson et al., Human Molecular Genetics, 2(8):1299-1302, 1993; Duff and Lincoln, “Insertion of a pathogenic mutationinto a yeast artificial chromosome containing the human APP gene andexpression in ES cells”, Research Advances in Alzheimer's Disease andRelated Disorders, 1995; Huxley et al., Genomics, 9:742-750 1991;Jakobovits et al., Nature, 362:255-261, 1993; Lamb et al., NatureGenetics, 5: 22-29, 1993; Pearson and Choi, Proc. Natl. Acad. Sci. USA,1993. 90:10578-82; Rothstein, Methods in Enzymology, 194:281-301, 1991;Schedl et al., Nature, 362: 258-261, 1993; Strauss et al., Science,259:1904-1907, 1993, all of which are incorporated herein by reference.

Of particular interest is the paper by Pascolo et al., published in J.Exp. Med. 185: 2043-2051, 1997, which describe the preparation of miceexpressing the human HLA-A2.1, H-2 Db and HHD MHC class I molecules anddevoid of mice MHC class I altogether.

An exemplary antibody, referred to as the H9 antibody, capable ofbinding to an MHC class I complexed with a CMV pp65 epitope comprisescomplementarity determining region (CDR) amino acid sequences as setforth in SEQ ID NOs:24-26 (for the heavy chain) and 30-32 (for the lightchain).

Another exemplary antibody, referred to as the F5 antibody, capable ofbinding to an MHC class I complexed with a CMV pp65 epitope comprisescomplementarity determining region (CDR) amino acid sequences as setforth in SEQ ID NOs:36-38 (for the heavy chain) and 42-44 (for the lightchain).

The invention provides a nucleic acid construct comprising a nucleicacid sequence encoding the CDR sequences of the heavy chain and thelight chain of the antibody of the invention. The nucleic acid constructmay further comprise a promoter for directing expression of the nucleicacid sequence in a host cell.

According to some embodiments of the invention, the nucleic acidconstruct comprising the nucleic acid sequences set forth by SEQ IDNOs:27-29 (for the heavy chain CDRs) and SEQ ID NOs:33-35 (for the lightchain CDRs).

According to some embodiments of the invention, the nucleic acidconstruct comprising the nucleic acid sequences set forth by SEQ IDNOs:39-41 (for the heavy chain CDRs) and SEQ ID NOs:45-47 (for the lightchain CDRs).

According to some embodiments of the invention, the nucleic acidconstruct comprising the nucleic acid sequence set forth by SEQ ID NO:17(for the heavy chain) and SEQ ID NO:19 (for the light chain).

According to some embodiments of the invention, the nucleic acidconstruct comprising the nucleic acid sequence set forth by SEQ ID NO:21(for the heavy chain) and SEQ ID NO:23 (for the light chain).

As mentioned herein above, the antibodies of the invention may beantibody fragments. Antibody fragments according to the invention can beprepared by proteolytic hydrolysis of the antibody or by expression inE. coli or mammalian cells (e.g. Chinese hamster ovary cell culture orother protein expression systems) of a DNA sequence encoding thefragment.

Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959.Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. According to some embodiments ofthe invention, the Fv fragments comprise V_(H) and V_(L) chainsconnected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

According to some embodiments of the invention, the antibodies aremultivalent forms such as tetrameric Fabs or IgG1 antibodies. Theadvantages of the multivalent forms of the antibody of the inventioninclude increased avidity, yet without compromising the antibodyspecificity to its target (i.e., the MHC-CMV pp65 peptide complex).Exemplary methods for generating tetrameric Fabs or IgG1 antibodies aredescribed in the general materials and experimental methods of theExamples section herein below.

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence.

The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

It will be appreciated that once the CDRs of an antibody are identified,using conventional genetic engineering techniques, expressiblepolynucleotides encoding any of the forms or fragments of antibodiesdescribed herein can be devised and modified in one of many ways inorder to produce a spectrum of related-products as further describedherein below.

The antibody of the invention can be used in vitro, ex vivo and in vivoin various therapeutic or diagnostic applications.

In case the antibody of the invention is to be used for administrationinto an individual (e.g., human), the human or humanized antibody orantibody fragment will generally tend to be better toleratedimmunologically than one of non human origin since non variable portionsof non human antibodies will tend to trigger xenogeneic immune responsesmore potent than the allogeneic immune responses triggered by humanantibodies which will typically be allogeneic with the individual. Itwill be preferable to minimize such immune responses since these willtend to shorten the half-life, and hence the effectiveness, of theantibody of the invention in the individual. Furthermore, such immuneresponses may be pathogenic to the individual, for example by triggeringharmful inflammatory reactions.

Alternately, an antibody or antibody fragment of human origin, or ahumanized antibody, will also be advantageous for applications in whicha functional physiological effect, for example an immune responseagainst a target cell, activated by a constant region of the antibody orantibody fragment in the individual is desired. For example, forapplications including targeted cell killing a specific immune responseis advantageous. Such applications particularly include those in whichthe functional interaction between a functional portion of the antibodyor antibody fragment, such as an Fc region, with a molecule such as anFc receptor or an Fc-binding complement component, is optimal when sucha functional portion is, similarly to the Fc region, of human origin.

Depending on the application and purpose, the antibody of the inventionwhich includes a constant region, or a portion thereof, of any ofvarious isotypes may be employed. According to some embodiments of theinvention, the isotype is selected so as to enable or inhibit a desiredphysiological effect, or to inhibit an undesired specific binding of theantibody of the invention via the constant region or portion thereof.For example, for inducing antibody-dependent cell mediated cytotoxicity(ADCC) by a natural killer (NK) cell, the isotype can be IgG; forinducing ADCC by a mast cell/basophil, the isotype can be IgE; and forinducing ADCC by an eosinophil, the isotype can be IgE or IgA. Forinducing a complement cascade the composition-of-matter may comprise anantibody or antibody fragment comprising a constant region or portionthereof capable of initiating the cascade. For example, the antibody orantibody fragment may advantageously comprise a Cgamma2 domain of IgG orCmu3 domain of IgM to trigger a C1q-mediated complement cascade.

Conversely, for avoiding an immune response, such as the aforementionedone, or for avoiding a specific binding via the constant region orportion thereof, the antibody of the invention may not comprise aconstant region (be devoid of a constant region), or a portion thereof,of the relevant isotype.

Additionally or alternatively, depending on the application and purpose,the antibody or antibody fragment may be attached to any of variousfunctional moieties. An antibody or antibody fragment, such as that ofthe invention, attached to a functional moiety may be referred to in theart as an “immunoconjugate”.

According to some embodiments of the invention, the functional moiety isa detectable moiety or a toxin. An antibody or antibody fragmentattached to a toxin may be referred to in the art as an “immunotoxin”.

As is described and demonstrated in further detail hereinbelow, adetectable moiety or a toxin may be particularly advantageously employedin applications of the invention involving use of the antibody of theinvention to detect the complex or cells expressing the complex of theMHC molecule and the cytomegalovirus (CMV) pp65 peptide and/or to killcells expressing or presenting such a complex.

For applications involving using the antibody of the invention to detectthe antigen-presenting portion of the complex, the detectable moietyattached to the antibody or antibody fragment can be a reporter moietyenabling specific detection of the MHC-CMV pp65 peptide complex bound bythe antibody or antibody fragment of the invention.

While various types of reporter moieties may be utilized to detect theMHC-CMV pp65 peptide complex, depending on the application and purpose,the reporter moiety can be a fluorophore or an enzyme. Alternately, thereporter moiety may be a radioisotope, such as [125]iodine. Furtherexamples of reporter moieties, including those detectable by PositronEmission Tomagraphy (PET) and Magnetic Resonance Imaging (MRI), are wellknown to those of skill in the art.

A fluorophore may be advantageously employed as a detection moietyenabling detection of the MHC-CMV pp65 peptide complex via any ofnumerous fluorescence detection methods. Depending on the applicationand purpose, such fluorescence detection methods include, but are notlimited to, fluorescence activated flow cytometry (FACS),immunofluorescence confocal microscopy, fluorescence in-situhybridization (FISH), fluorescence resonance energy transfer (FRET), andthe like.

Various types of fluorophores, depending on the application and purpose,may be employed to detect the MHC-CMV pp65 peptide complex.

Examples of suitable fluorophores include, but are not limited to,phycoerythrin (PE), fluorescein isothiocyanate (FITC), Cy-chrome,rhodamine, green fluorescent protein (GFP), blue fluorescent protein(BFP), Texas red, PE-Cy5, and the like.

Preferably, the fluorophore is phycoerythrin.

As is described and illustrated in the Examples section below, theantibody of the invention attached to a fluorophore, such asphycoerythrin, can be used to optimally detect the MHC-CMV pp65 peptidecomplex using various immunofluorescence-based detection methods.

Ample guidance regarding fluorophore selection, methods of linkingfluorophores to various types of molecules, such as an antibody orantibody fragment of the invention, and methods of using such conjugatesto detect molecules which are capable of being specifically bound byantibodies or antibody fragments comprised in such immunoconjugates isavailable in the literature of the art [for example, refer to: RichardP. Haugland, “Molecular Probes: Handbook of Fluorescent Probes andResearch Chemicals 1992-1994”, 5th ed., Molecular Probes, Inc. (1994);U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.; Hermanson, “BioconjugateTechniques”, Academic Press New York, N.Y. (1995); Kay M. et al., 1995.Biochemistry 34:293; Stubbs et al., 1996. Biochemistry 35:937; GakamskyD. et al., “Evaluating Receptor Stoichiometry by Fluorescence ResonanceEnergy Transfer,” in “Receptors: A Practical Approach,” 2nd ed.,Stanford C. and Horton R. (eds.), Oxford University Press, UK. (2001);U.S. Pat. No. 6,350,466 to Targesome, Inc.]. While various methodologiesmay be employed to detect the MHC-CMV pp65 peptide complex using afluorophore, such detection is preferably effected as described anddemonstrated in the Examples section below.

Alternately, an enzyme may be advantageously utilized as the detectablemoiety to enable detection of the antigen-presenting portion of thecomplex via any of various enzyme-based detection methods. Examples ofsuch methods include, but are not limited to, enzyme linkedimmunosorbent assay (ELISA; for example, to detect theantigen-presenting portion of the complex in a solution), enzyme-linkedchemiluminescence assay (for example, to detect the complex in anelectrophoretically separated protein mixture), and enzyme-linkedimmunohistochemical assay (for example, to detect the complex in a fixedtissue).

Numerous types of enzymes may be employed to detect theantigen-presenting portion of the complex, depending on the applicationand purpose. For example, an antibody or antibody fragment attached toan enzyme such as horseradish peroxidase can be used to effectivelydetect the MHC-CMV pp65 peptide complex, such as via ELISA, orenzyme-linked immunohistochemical assay.

Examples of suitable enzymes include, but are not limited to,horseradish peroxidase (HPR), beta-galactosidase, and alkalinephosphatase (AP).

Ample guidance for practicing such enzyme-based detection methods isprovided in the literature of the art (for example, refer to: KhatkhatayM I. and Desai M., 1999. J Immunoassay 20:151-83; Wisdom G B., 1994.Methods Mol. Biol. 32:433-40; Ishikawa E. et al., 1983. J Immunoassay4:209-327; Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208;Schuurs A H. and van Weemen B K., 1980. J Immunoassay 1:229-49). Whilevarious methodologies may be employed to detect the antigen-presentingportion of the complex using an enzyme, such detection is preferablyeffected as described in the Examples section below.

The functional moiety may be attached to the antibody or antibodyfragment in various ways, depending on the context, application andpurpose.

A polypeptidic functional moiety, in particular a polypeptidic toxin,may be advantageously attached to the antibody or antibody fragment viastandard recombinant techniques broadly practiced in the art (forExample, refer to Sambrook et al., infra, and associated references,listed in the Examples section which follows).

A functional moiety may also be attached to the antibody or antibodyfragment using standard chemical synthesis techniques widely practicedin the art [for example, refer to the extensive guidelines provided byThe American Chemical Society (for example at:hypertexttransferprotocol://worldwideweb (dot) chemistry (dot)org/portal/Chemistry)]. One of ordinary skill in the art, such as achemist, will possess the required expertise for suitably practicingsuch chemical synthesis techniques.

Alternatively, a functional moiety may be attached to the antibody orantibody fragment by attaching an affinity tag-coupled antibody orantibody fragment of the invention to the functional moiety conjugatedto a specific ligand of the affinity tag.

Various types of affinity tags may be employed to attach the antibody orantibody fragment to the functional moiety.

Examples of detectable moieties that can be used in the inventioninclude but are not limited to radioactive isotopes, phosphorescentchemicals, chemiluminescent chemicals, fluorescent chemicals, enzymes,fluorescent polypeptides and epitope tags. The detectable moiety can bea member of a binding pair, which is identifiable via its interactionwith an additional member of the binding pair, and a label which isdirectly visualized. In one example, the member of the binding pair isan antigen which is identified by a corresponding labeled antibody. Inone example, the label is a fluorescent protein or an enzyme producing acolorimetric reaction.

When the detectable moiety is a polypeptide, the immunolabel (i.e. theantibody conjugated to the detectable moiety) may be produced byrecombinant means or may be chemically synthesized by, for example, thestepwise addition of one or more amino acid residues in defined orderusing solid phase peptide synthetic techniques. Examples of polypeptidedetectable moieties that can be linked to the antibodies of theinvention using recombinant DNA technology include fluorescentpolypeptides, phosphorescent polypeptides, enzymes and epitope tags.

Expression vectors can be designed to fuse proteins encoded by theheterologous nucleic acid insert to fluorescent polypeptides. Forexample, antibodies can be expressed from an expression vector fusedwith a green fluorescent protein (GFP)-like polypeptide. A wide varietyof vectors are commercially available that fuse proteins encoded byheterologous nucleic acids to the green fluorescent protein fromAequorea victoria (“GFP”), the yellow fluorescent protein and the redfluorescent protein and their variants (e.g., Evrogen). In thesesystems, the fluorescent polypeptide is entirely encoded by its aminoacid sequence and can fluoresce without requirement for cofactor orsubstrate. Expression vectors that can be employed to fuse proteinsencoded by the heterologous nucleic acid insert to epitope tags arecommercially available (e.g., BD Biosciences, Clontech).

Alternatively, chemical attachment of a detectable moiety to theantibodies of the invention can be effected using any suitable chemicallinkage, direct or indirect, as via a peptide bond (when the detectablemoiety is a polypeptide), or via covalent bonding to an interveninglinker element, such as a linker peptide or other chemical moiety, suchas an organic polymer. Such chimeric peptides may be linked via bondingat the carboxy (C) or amino (N) termini of the peptides, or via bondingto internal chemical groups such as straight, branched or cyclic sidechains, internal carbon or nitrogen atoms, and the like. Such modifiedpeptides can be easily identified and prepared by one of ordinary skillin the art, using well known methods of peptide synthesis and/orcovalent linkage of peptides. Description of fluorescent labeling ofantibodies is provided in details in U.S. Pat. Nos. 3,940,475,4,289,747, and 4,376,110.

Exemplary methods for conjugating two peptide moieties are describedherein below:

SPDP Conjugation:

Any SPDP conjugation method known to those skilled in the art can beused. For example, in one illustrative embodiment, a modification of themethod of Cumber et al. (1985, Methods of Enzymology 112: 207-224) asdescribed below, is used.

A peptide, such as an identifiable or therapeutic moiety, (1.7 mg/ml) ismixed with a 10-fold excess of SPDP (50 mM in ethanol) and the antibodyis mixed with a 25-fold excess of SPDP in 20 mM sodium phosphate, 0.10 MNaCl pH 7.2 and each of the reactions incubated, e.g., for 3 hours atroom temperature. The reactions are then dialyzed against PBS.

The peptide is reduced, e.g., with 50 mM DTT for 1 hour at roomtemperature. The reduced peptide is desalted by equilibration on G-25column (up to 5% sample/column volume) with 50 mM KH₂PO₄ pH 6.5. Thereduced peptide is combined with the SPDP-antibody in a molar ratio of1:10 antibody:peptide and incubated at 4° C. overnight to form apeptide-antibody conjugate.

Glutaraldehyde Conjugation:

Conjugation of a peptide (e.g., an identifiable or therapeutic moiety)with an antibody can be accomplished by methods known to those skilledin the art using glutaraldehyde. For example, in one illustrativeembodiment, the method of conjugation by G. T. Hermanson (1996,“Antibody Modification and Conjugation, in Bioconjugate Techniques,Academic Press, San Diego) described below, is used.

The antibody and the peptide (1.1 mg/ml) are mixed at a 10-fold excesswith 0.05% glutaraldehyde in 0.1 M phosphate, 0.15 M NaCl pH 6.8, andallowed to react for 2 hours at room temperature. 0.01 M lysine can beadded to block excess sites. After-the reaction, the excessglutaraldehyde is removed using a G-25 column equilibrated with PBS (10%v/v sample/column volumes)

Carbodiimide Conjugation:

Conjugation of a peptide with an antibody can be accomplished by methodsknown to those skilled in the art using a dehydrating agent such as acarbodiimide. Most preferably the carbodiimide is used in the presenceof 4-dimethyl aminopyridine. As is well known to those skilled in theart, carbodiimide conjugation can be used to form a covalent bondbetween a carboxyl group of peptide and an hydroxyl group of an antibody(resulting in the formation of an ester bond), or an amino group of anantibody (resulting in the formation of an amide bond) or a sulfhydrylgroup of an antibody (resulting in the formation of a thioester bond).

Likewise, carbodiimide coupling can be used to form analogous covalentbonds between a carbon group of an antibody and an hydroxyl, amino orsulfhydryl group of the peptide. See, generally, J. March, AdvancedOrganic Chemistry: Reaction's, Mechanism, and Structure, pp. 349-50 &372-74 (3d ed.), 1985. By means of illustration, and not limitation, thepeptide is conjugated to an antibody via a covalent bond using acarbodiimide, such as dicyclohexylcarbodiimide. See generally, themethods of conjugation by B. Neises et al. (1978, Angew Chem., Int. Ed.Engl. 17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); E. P.Boden et al. (1986, J. Org. Chem. 50:2394) and L. J. Mathias (1979,Synthesis 561). The level of immunocomplex may be compared to a controlsample from a non-diseased subject, wherein an up-regulation ofimmunocomplex formation is indicative of disease associated with CMVinfection. Preferably, the subject is of the same species e.g. human,preferably matched with the same age, weight, sex etc. It will beappreciated that the control sample may also be of the same subject froma healthy tissue, prior to disease progression or following diseaseremission.

Preferably, the affinity tag is a biotin molecule, more preferably astreptavidin molecule.

A biotin or streptavidin affinity tag can be used to optimally enableattachment of a streptavidin-conjugated or a biotin-conjugatedfunctional moiety, respectively, to the antibody or antibody fragmentdue to the capability of streptavidin and biotin to bind to each otherwith the highest non covalent binding affinity (i.e., with a Kd of about10⁻¹⁴ to 10⁻¹⁵). A biotin affinity tag may be highly advantageous forapplications benefiting from. Thus, the antibody of invention can be amultimeric form of the antibody or antibody fragment, which may beoptimally formed by conjugating multiple biotin-attached antibodies orantibody fragments of the invention to a streptavidin molecule, asdescribed in further detail below.

As used herein the term “about” refers to plus or minus 10 percent.

Various methods, widely practiced in the art, may be employed to attacha streptavidin or biotin molecule to a molecule such as the antibody orantibody fragment to a functional moiety.

For example, a biotin molecule may be advantageously attached to anantibody or antibody fragment of the invention attached to a recognitionsequence of a biotin protein ligase. Such a recognition sequence is aspecific polypeptide sequence serving as a specific biotinylationsubstrate for the biotin protein ligase enzyme. Ample guidance forbiotinylating a target polypeptide such as an antibody fragment using arecognition sequence of a biotin protein ligase, such as the recognitionsequence of the biotin protein ligase BirA, is provided in theliterature of the art (for example, refer to: Denkberg, G. et al., 2000.Eur. J. Immunol. 30:3522-3532). Preferably, such biotinylation of theantibody or antibody fragment is effected as described and illustratedin the Examples section below.

Alternately, various widely practiced methods may be employed to attacha streptavidin molecule to an antibody fragment, such as a single chainFv (for example refer to Cloutier S M. et al., 2000. MolecularImmunology 37:1067-1077; Dubel S. et al., 1995. J Immunol Methods178:201; Huston J S. et al., 1991. Methods in Enzymology 203:46;Kipriyanov S M. et al., 1995. Hum Antibodies Hybridomas 6:93; KipriyanovS M. et al., 1996. Protein Engineering 9:203; Pearce L A. et al., 1997.Biochem Molec Biol Intl 42:1179-1188).

Functional moieties, such as fluorophores, conjugated to streptavidinare commercially available from essentially all major suppliers ofimmunofluorescence flow cytometry reagents (for example, Pharmingen orBecton-Dickinson). Standard recombinant DNA chemical techniques arepreferably employed to produce a fusion protein comprising streptavidinfused to a polypeptidic functional moiety. Standard chemical synthesistechniques may also be employed to form the streptavidin-functionalmoiety conjugate. Extensive literature is available providing guidancefor the expression, purification and uses of streptavidin orstreptavidin-derived molecules (Wu S C. et al., 2002. Protein Expressionand Purification 24:348-356; Gallizia A. et al., 1998. ProteinExpression and Purification 14:192-196), fusion proteins comprisingstreptavidin or streptavidin-derived molecules (Sano T. and Cantor C R.,2000. Methods Enzymol. 326:305-11), and modified streptavidin orstreptavidin-derived molecules (see, for example: Sano T. et al., 1993.Journal of Biological Chemistry 270:28204-28209), including forstreptavidin or streptavidin-derived molecules whose gene sequence hasbeen optimized for expression in E. coli (Thompson L D. and Weber P C.,1993. Gene 136:243-6).

As mentioned, the antibody may be conjugated to a therapeutic moiety.The therapeutic moiety can be, for example, a cytotoxic moiety, a toxicmoiety, a cytokine moiety and a second antibody moiety comprising adifferent specificity to the antibodies of the invention.

In a similar fashion to an immunolabel, an immunotoxin (i.e. atherapeutic moiety attached to an antibody of the invention) may begenerated by recombinant or non-recombinant means. Thus, the inventionenvisages a first and second polynucleotide encoding the antibody of theinvention and the therapeutic moiety, respectively, ligated in frame, soas to encode an immunotoxin. The following Table 1 provides examples ofsequences of therapeutic moieties.

TABLE 1 Amino Acid sequence Nucleic Acid sequence (Genbank Accession(Genbank Accession Therapeutic Moiety No.) No.) Pseudomonas exotoxinAAB25018 S53109 Diphtheria toxin E00489 E00489 interleukin 2 CAA00227A02159 CD3 P07766 X03884 CD16 AAK54251 AF372455 interleukin 4 P20096ICRT4 HLA-A2 P01892 K02883 interleukin 10 P22301 M57627 Ricin A toxin225988 A23903

According to some embodiments of the invention, the toxic moiety is PE38KDEL.

Exemplary methods of conjugating the antibodies of the invention topeptide therapeutic agents are described herein above.

As mentioned, the antibody of the invention, which is capable ofspecifically recognizing and binding an MHC-CMV pp65 peptide complex asdescribed above, can be used to detecting cell expressing acytomegalovirus (CMV) antigen.

Thus, according to an aspect of some embodiments of the invention thereis provided a method of detecting a cell expressing a cytomegalovirus(CMV) antigen. The method is effected by contacting the cell with theantibody under conditions which allow immunocomplex formation, wherein apresence or a level above a predetermined threshold of the immunocomplexis indicative of CMV expression in the cell.

The contacting may be effected in vitro (e.g., in a cell line), ex vivoor in vivo.

As mentioned, the method of the invention is effected under conditionssufficient to form an immunocomplex (e.g. a complex between theantibodies of the invention and the MHC-CMV pp65 peptide); suchconditions (e.g., appropriate concentrations, buffers, temperatures,reaction times) as well as methods to optimize such conditions are knownto those skilled in the art, and examples are disclosed herein.

As described in the Examples section which follows, the immunocomplexcan be formed and detected within the cell or on the cell surface. Fordetection in the cell, the conditions include a permeabilization agent(e.g., a solution including saponin), to enable penetration of theantibody inside the cell. According to some embodiments of theinvention, the immunocomplex is formed on the surface of the cell.

Determining a presence or level of the immunocomplex of the invention isdependent on the detectable moiety to which the antibody is attached,essentially as described hereinabove.

A non-limiting example of the immunocomplex of the invention is thecomplex formed between the antibody of the invention (e.g., H9 or F5)and a protein complex comprising MHC class I heavy chain (HLA-A2) andpp65 peptide as set forth by SEQ ID NO:3.

As mentioned, the antibody of the invention, which is capable ofspecifically recognizing and binding an MHC-CMV pp65 peptide complex,can be used to diagnose CMV infection in a subject in need thereof.

Thus, according to another aspect of the invention, there is provided amethod of diagnosing a cytomegalovirus (CMV) infection in a subject inneed thereof. The method is effected by contacting a cell of the subjectwith the antibody under conditions which allow immunocomplex formation,wherein a presence or a level above a pre-determined threshold of theimmunocomplex in the cell is indicative of the CMV infection in thesubject.

As used herein the phrase “subject in need thereof” refers to a mammal,preferably, a human subject which is suspected of being infected withCMV.

According to some embodiments of the invention, the subject has asuppressed or a compromised immune system, such as an immuno-compromisedorgan transplant recipient or a subject infected with humanimmunodeficiency virus (HIV).

According to some embodiments of the invention, the CMV infection isassociated with a disease selected from the group consisting ofmononucleosis, retinitis, pneumonia, gastrointestinal disorders, andencephalitis.

According to some embodiments of the invention, the cell is a retinacell, lung epithelial cell, a gastrointestinal epithelial cell and/or abrain cell.

The antibody described herein can be used to treat a disease associatedwith CMV infection.

According to an additional aspect of the invention there is provided amethod of treating a disease associated with cytomegalovirus (CMV)infection, the method is effected by administering to a subject in needthereof a therapeutically effective amount of the antibody therebytreating the disease associated with CMV infection.

The term “treating” refers to inhibiting or arresting the development ofa disease, disorder or condition and/or causing the reduction,remission, or regression of a disease, disorder or condition. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a disease, disorder orcondition, and similarly, various methodologies and assays may be usedto assess the reduction, remission or regression of a disease, disorderor condition.

The antibodies of the invention may be provided per se or may beadministered as a pharmaceutical composition.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the antibodies of theinvention accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the invention may be manufactured byprocesses well known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the inventionthus may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the invention are conveniently delivered in the form of anaerosol spray presentation from a pressurized pack or a nebulizer withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in a dispenser may be formulated containing apowder mix of the compound and a suitable powder base such as lactose orstarch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the invention may also be formulatedin rectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the inventioninclude compositions wherein the active ingredients are contained in anamount effective to achieve the intended purpose. More specifically, atherapeutically effective amount means an amount of active ingredients(the antibody of the invention or the nucleic acid construct encodingsame) effective to prevent, alleviate or ameliorate symptoms of apathology, (e.g., a disease associated with cytomegalovirus infection)or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredient are sufficient to induceor suppress the biological effect (minimal effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the invention may, if desired, be presented in a pack ordispenser device, such as an FDA approved kit, which may contain one ormore unit dosage forms containing the active ingredient. The pack may,for example, comprise metal or plastic foil, such as a blister pack. Thepack or dispenser device may be accompanied by instructions foradministration and use. The pack or dispenser may also be accommodatedby a notice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

As used herein the term “about” refers to ±10%.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

General Materials and Experimental Methods Generation of BiotinylatedSingle-Chain MHC/Peptide Complexes

Single-chain MHC (scMHC)/peptide complexes were produced by in vitrorefolding of inclusion bodies produced in Escherichia coli, as described(Denkberg, G. et al., 2000). Briefly, a single-chain β₂-microglobulin(β₂m)-HLA/A2 (scMHC) construct, in which the β₂m and HLA-A2 genes areconnected to each other by a flexible peptide linker (wherein the β2mgene is translationally fused upstream of the gene encoding the MHCheavy chain) (HLA-A2), was engineered to contain the BirA recognitionsequence for site-specific biotinylation at the C terminus (scMHC-BirA).In vitro refolding was performed in the presence of a 5-10 molar excessof the antigenic peptides, as described (Denkberg, G. et al., 2000).Correctly folded MHC/peptide complexes were isolated and purified byanion exchange Q-Sepharose chromatography (Pharmacia, Peapack, N.J.),followed by site-specific biotinylation using the BirA enzyme (Avidity,Denver, Colo.), as previously described (Altman, J. D. et al., 1996).The homogeneity and purity of the scMHC-peptide complexes were analyzedby various biochemical means, including SDS-PAGE, size exclusionchromatography, and ELISA, as previously described (Denkberg, G. et al.,2000).

Selection of Phage Antibodies on Biotinylated Complexes

Selection of phage Abs on biotinylated complexes was preformed, asdescribed (Denkberg, G., et al., 2002; Lev A., et al., 2002). Briefly, alarge human Fab library containing 3.7×10¹⁰ different Fab clones (DeHaard, H J., et al., 1999) was used for the selection. Phages (10¹³)were first preincubated with streptavidin-coated paramagnetic beads (200μl; Dynal, Oslo, Norway) to deplete the streptavidin binders. Theremaining phages were subsequently used for panning with decreasingamounts of biotinylated scMHC-peptide complexes. Thestreptavidin-depleted library was incubated in solution with solublebiotinylated scHLA-A2/pp65 complexes (500 nM for the first round, and100 nM for the following rounds) for 30 minutes at room temperature(RT).

Streptavidin-coated magnetic beads (200 μl for the first round ofselection, and 100 μl for the second and third rounds) were added to themixture and incubated for 10-15 minutes at RT. The beads were washedextensively 12 times with PBS/Tween 0.1%, and additional two washes werewith PBS. Bound phages were eluted with triethylamine (100 mM, 5 minutesat RT), followed by neutralization with Tris-HCl (1 M, pH 7.4), and usedto infect E. coli TG1 cells (OD=0.5) for 30 minutes at 37° C.

The diversity of the selected Abs was determined by DNA fingerprintingusing a restriction endonuclease (BstNI), which is a frequent cutter ofAb V gene sequences. The Fab DNA of different clones was PCR amplifiedusing the primers pUC-reverse [5′-AGCGGATAACAATTTCACACAGG-3′ (SEQ IDNO:1)] and fd-tet-seq24 [5′-TTTGTCGTCTTTCCAGACGTTAGT-3′ (SEQ ID NO:2)],followed by digestion with BstNI (NEB, Beverly, Mass.) (2 hours, 60° C.)and analysis on agarose gel electrophoresis.

Expression and Purification of Soluble Recombinant Fab Abs

Fab Abs were expressed and purified, as described recently (Denkberg,G., et al., 2000). BL21 bacterial cells were grown to OD₆₀₀=0.8-1.0 andinduced to express the recombinant Fab Ab by the addition of 1 mMisopropyl 13-D-thiogalactoside (IPTG) for 3-4 hours at 30° C.Periplasmic content was released using the B-PER solution (Pierce,Rockford, Ill.), which was applied onto a prewashed TALON column(Clontech, Palo Alto, Calif.). Bound Fabs were eluted using 0.5 ml of100 mM imidazole in PBS. The eluted Fabs were dialyzed twice against PBS(overnight, 4° C.) to remove residual imidazole.

ELISA with Phage Clones and Purified Fab Abs

The binding specificities of individual phage clones and soluble Fabwere determined by ELISA using biotinylated scMHC-peptide complexes.ELISA plates (Falcon) were coated overnight with BSA-biotin (1 μg/well).After having been washed, the plates were incubated (1 hour, RT) withstreptavidin (1 μg/well), washed extensively, and further incubated (1hour, RT) with 0.5 μg of MHC/peptide complexes. The plates were blockedfor 30 minutes at RT with PBS/2% skim milk and subsequently wereincubated for 1 hour at RT with phage clones (˜10⁹ phages/well) orvarious concentrations of soluble purified Fab. After having beenwashed, the plates were incubated with HRP-conjugated/anti-human Fab Ab(for soluble Fabs) or HRP-conjugated anti-M13 phage (for phage-displayedFabs). Detection was performed using tetramethylbenzidine reagent(Sigma-Aldrich, St. Louis, Mo.). The HLA-A2-restricted peptides used forspecificity studies of the Fab phage clones or purified Fab Abs are asdescribed in Examples 1 and 2 below.

Generation of Fluorescently-Labeled Tetrameric Fab

The genes encoding the L and H chain of Fab H9 were cloned separatelyinto a T7-promotor pET-based expression vector. The L chain gene wasengineered to contain the BirA recognition sequence for site-specificbiotinylation at the C terminus. These constructs were expressedseparately in E. coli BL21 cells and upon induction with IPTG,intracellular inclusion bodies that contain large amounts of therecombinant protein accumulated. Inclusion bodies of both chains werepurified, solubilized, reduced with 10 mg/ml DTE (Dithioerithrol), andsubsequently refolded at a 1:1 ratio in a redox-shuffling buffer systemcontaining 0.1 M Tris, 0.5 M arginine, and 0.09 mM oxidized glutathione,pH 8.0. Correctly folded Fab was then isolated and purified by anionexchange MonoQ chromatography (Pharmacia). The Fab peak fractions wereconcentrated using Centricon-30 (Amicon, Beverly, Mass.) to 1 mg/ml, andthe buffer was exchanged to Tris-HCl (10 mM, pH 8.0). Biotinylation wasperformed using the BirA enzyme (Avidity), as previously described.Excess biotin was removed from biotinylated Fabs using a G-25 desaltingcolumn. PE-labeled streptavidin (Jackson ImmunoResearch, West Grove,Pa.) was added at a molar ratio of 1:4 to produce fluorescent tetramersof the biotinylated Fab.

Generation of Whole IgG from Recombinant Fab

To transform the recombinant fragments into whole IgG molecules, theeukaryotic expression vector pCMV/myc/ER (Invitrogen) was used. Theheavy and the light chains of the Fab were cloned separately. Eachshuttle expression vector carries a different antibiotics resistancegene and thus expression was facilitated by co-transfection of the twoconstructs into human embryonic kidney HEK293 cells. Cotransfections ofHEK293 cells were performed using the nonliposomal transfection reagentFuGene 6 (Roche, Brussels, Belgium) according to the manufacturer'sinstructions. The transfection was performed with serum free mediumcontaining 0.8 mg/ml of G418, and 100 μg/ml of hygromycin. Forty-eighthours after transfection limiting dilutions were performed into mediumcontaining 0.8 mg/ml of G418, and 100 μg/ml of hygromycin. Cells wereplated in 96-well plates at 1000 cells per well. Medium was exchangedafter 5 and 10 days. Wells in which a single colony grew up to 50% ofthe well were further trypsinized with 20 μl and 20 μl medium andsplitted into two wells: 10 μl into a 24 well plate (backup) and 30 μlinto a 24 well plate (experiment). When the plate reached 80%confluency, serum starvation was initiated by reducing each day serumpercentile to 0.5%. After 48 hours of incubation with 0.5% fetal calfserum (FCS), screening of cell culture supernatants was performed byELISA and FACS assays. The IgG secreting clones that exhibited the bestbinding reactivity as detected by ELISA, FACS and the highest amount ofprotein, were selected for antibody production and purification. ProteinA-Sepharose™ 4 Fast Flow beads (Amersham) were prepared according to themanufacturer's instructions. Briefly, supernatant was loaded on theProtein A-Sepharose beads at 15-50 ml/h. Unbound immunoglobulins werewashed with 0.001 M NaH₂PO₄ and 0.019 M Na₂HPO₄. Bound immunoglobulinswere then eluted with 0.1 M citric acid at pH 3. Five fractions werecollected with 250 μl of elusion buffer and immediately neutralized with80 μl of Tris-HCL pH 9. IgG concentration was measured using the Pierceprotein assay. The eluted protein was dialyzed against PBS pH 7.4 overnight. 10 mgs of IgG were produced from 1 L of culture supernatant.

Flow Cytometry

The B cell line RMAS-HHD, which is transfected with a single-chainβ₂m-HLA-A2 gene, the EBV-transformed HLA-A2⁺ JY cells, and the HLA-A2-Bcell line APD-70 were used to determine the reactivity of therecombinant Fab Abs with cell surface-expressed HLA-A2/peptidecomplexes. Peptide pulsing was performed as indicated: 10⁶ cells werewashed twice with serum-free RPMI and incubated overnight at 26° C. or37° C., respectively, in medium containing 1-50 μM of the peptide. TheRMAS-HHD cells were subsequently incubated at 37° C. for 2-3 hours tostabilize cell surface expression of MHC-peptide complexes.

Cells were incubated for 60 minutes at 4° C. with recombinant Fab Abs(10 μg/ml) in 100 μl PBS. After one wash, the cells were incubated with1 μg anti-human Fab (Jackson ImmunoResearch) for another 60 minutes at4° C. After three washes, the cells were resuspended in ice-cold PBS.The cells were analyzed by a FACStar flow cytometer (BD Biosciences, SanJose, Calif.).

Surface Plasmon Resonance

0.0025 mg/ml of biotinylated HLA-A2/pp65 or control HLA-A2/EBV complexeswere bound to a streptavidin (SA) sensor chip (Biacore, Uppsala, Sweden)per well. Measurements of 780-800 RU were detected for each well aftercomplexes binding. Soluble isolated antibodies in their monomeric/IgGform were diluted in PBS at three concentration (0.05 μM, 0.1 μM, 0.2μM) and were flowed over the relevant wells at a rate of 10 μl/min atroom temperature. Responses were recorded using Biacore 2000 andanalyzed using BIAevaluation software 3.2 (Biacore, Uppsala, Sweden).

Cell Infection

Human fibroblasts which express the HLA-A2 allele were obtained fromprimary cultures of foreskins and grown in Dulbecco's modified Eagle'smedium (DMEM), containing 2 mM Glutamine, 100 IU of penicillin/ml, 10%fetal calf serum (FCS), non essential amino acid (1:100), sodiumPyruvate (1:100) and 10 mM hepes. The cells were infected at an MOI of0.5-1 with the laboratory strain AD169⁴¹ and harvested at five timescales for FACS analysis. MHC expression on virus infected or uninfectedcells was determined using PE conjugated anti HLA-A2 (BB7.2) monoclonalantibody. Detection of infection was with anti pp65 monoclonal antibody(clone IL11, Virusys, Sykesville, Md. USA) and anti mouse PE assecondary antibody. For intracellular staining cells were fixed with0.3% formaldehyde and then permeabilized with PBS containing 0.05%Saponin and 1% goat serum used for blocking.

Cytotoxicity Assay

Target cells were cultured in 48-well plates in DMEM medium plus 10% FCSand were grown up until confluent. Cells were washed and incubatedovernight with 15 μCi/ml (1Ci 37 GBq) [35^(S)] methionine (NEN). After 1hour of incubation with the IgG H9 (10-20 μg/ml or the indicatedconcentration at 37° C.), effector CTL cells were added at atarget:effector ratio of 1:3 respectively and incubated for 5 hours at37° C. After incubation, [35^(S)] methionine release from target cellswas measured in a 50-n1 sample of the culture supernatant. All assayswere performed in triplicate.

Confocal Microscopy

Infected and noninfected fibroblast cells were fixed for 10 minutes with0.5% paraformaldehyde, and washed twice with PBS containing 0.1% bovineserum albumin (BSA). The cells were permeabilized and incubated withanti pp65 mAb, H9 IgG, anti calnexin (Chemicon, cat. No. MAB3126),and/or cis-golgi matrix protein (GM130) (BD transduction laboratories,cat No. 610822) in the presence of a PBS medium containing 0.05%saponin, 1% fetal bovine serum, and 0.1% BSA, for 40 minutes at 4° C.Cells were subsequently washed and further incubated with goat antimouse secondary Ab conjugated to Alexa-flour⁵⁹⁴ (Molecular Probes, cat.No. A21216), and goat anti human secondary Ab conjugated toAlexa-flour⁴⁸⁸ (Molecular Probes, cat. No. A11013), respectively. DRAQ5(Alexis Biochemicals) was added to the stained cells before they werewashed again. Images were collected on a LSM 510 META laser scanningmicroscope (Carl Zeiss Microimaging Inc) using a x63 oil immersionobjective numerical aperture 1.32, at different zoom factors. AlexaFluor⁴⁸⁸ was excited using an argon laser at 488 nm. Alexa Fluor⁵⁹⁴ wasexcited using a krypton laser at 568 mm Differential interferencecontrast images were collected simultaneous with the fluorescence imagesusing the transmitted light detector. Z stacks of images were collectedusing a step increment of 0.3 μm between planes. All pictures were takenwith identical settings.

Isolation of PBMCs

Samples of 20-30 ml blood obtained from healthy donors or BMT patients,containing 500 units (U) of heparin was added to 50 ml sterile tubescontaining 15 ml Lymphoprep™ (Axis shield PoC AS, Oslo Norway). Theblood was added gently without mixing between the Ficoll and the blood.The tubes were centrifuged for 30 minutes at 1000 g without brakes. Theupper layer that contains the serum was removed and the Buffy coat thatcontains the peripheral blood mononuclear cells (PBMCs) was transferredto new tubes. The PBMCs were washed twice with 40 ml of phosphate buffersaline (PBS) and 2 mM EDTA (centrifuged at 700 g for 8 minutes). ThePBMCs were resuspended in 20 ml PBS, counted, centrifuged at 500 g for 8minutes and resuspended in PBMCs medium at 1-5×10⁶ cells/ml. About70×10⁶ cells are isolated from a total of 50 ml blood sample.

Example 1 Selection and Cloning of Recombinant Antibodies Specific forHLA-A2-PP65 Complex

Experimental Results

Selection of Recombinant Antibodies Specific for HLA-A2/pp65 Complexes

Recombinant peptide-HLA-A2 complexes that present the pp65₄₉₅₋₅₀₃ (SEQID NO:3) CMV-derived peptide were generated using a single-chain MHC(scMHC) construct according to the method previously describedpreviously (Denkberg G., et al., 2000). In this construct, theextracellular domains of HLA-A2 are connected into a single-chainmolecule with β₂m using a 15-aa flexible linker (the β2m istranslationally fused upstream of the MHC heavy chain). ThescMHC-peptide complexes were produced by in vitro refolding of inclusionbodies in the presence of the pp65 495-503 peptide (SEQ ID NO:3). Therefolded scHLA-A2/pp65 complexes were found to be pure, homogenous, andmonomeric by SDS-PAGE and size exclusion chromatography analyses (datanot shown). Recombinant scMHC-peptide complexes generated by thisstrategy were previously characterized in detail for their biochemical,biophysical, and biological properties, and were found to be correctlyfolded and functional (Denkberg G., et al., 2000; Denkberg G., et al.,2001).

A large human Fab library containing 3.7×10¹⁰ different Fab clones wasused for the selection on biotinylatd HLA-A2/pp65 complexes (De Haard HJ., et al., 1999). Phage displayed antibodies which were capable ofbinding to the specific biotinylated HLA-A2/peptide complex wereselected as previously described (Denkberg G., et al., 2002; Lev A., etal., 2002). Enrichment in phage titer was observed after three rounds ofpanning (Table 2, hereinbelow). Specificity of the selected phageantibodies against the complex was analyzed by a differential ELISAassay in which binding was tested against specific (pp65 495-503peptide; SEQ ID NO:3) and non specific (gp100 280-288 peptide; SEQ IDNO:4) biotinylated HLA-A2/peptide complexes. These were immobilized towells through BSA-biotin-streptavidin. As shown in FIG. 1 a, a highpercentage of specific clones was observed; 54 clones of the 96 screened(56%), were peptide specific and bound the specific peptide/MHC used inthe selection (i.e., the scHLA-A2/pp65 complex).

TABLE 2 Results of the amounts of phages counted before and after eachpanning (inputs and outputs). Enrichment of the outputs can be seen ineach panning round. Round of Panning Phage input Phage output Enrichment1^(st) 10¹² 4 × 10⁵ 2^(nd) 1.5 × 10¹²   5 × 10⁶  75 3^(rd) 5 × 10¹² 1.5× 10⁹   750

Cloning of Two Fab Clones with Specificity to the HLA-A2-pp65₄₉₅₋₅₀₃Complex

The diversity within the selected TCR-like Fabs was assessed by DNAfingerprint analysis using the BstNI restriction enzyme. The analysisrevealed two different clones, termed H9 and F5 with HLA-A2/pp65specificity (data not shown). DNA sequencing analysis confirmed theseobservations. The nucleic acid and amino acid sequences of the heavy andlight chains of H9Fab clone are provided in FIGS. 14 a-d (SEQ IDNOs:16-19). The nucleic acid and amino acid sequences of the heavy andlight chains of F5 Fab clone are provided in FIGS. 15 a-d (SEQ IDNOs:20-23). The amino acid sequences of the CDRs of the H9 and F5 FabAbs are provided in Table 3, hereinbelow. The nucleic acid sequences ofthe CDRs of the H9 and F5 Fab Abs are provided in Table 4, hereinbelow.

TABLE 3 Table 3: CDRs (amino acid sequences) of the heavy and lightchains of Fabs H9 and F5.Amino acid sequences of the CDRs of the Fab antibodies Fab cloneCDRs heavy chain CDRs light chain H9 SYAISW (SEQ ID NO: 24; CDR1)RASQSVSSSYLA (SEQ ID NO: 30; GIIPIFGTANYAQKFQG (SEQ ID NO: 25; CDR1)CDR2) GASSRAT (SEQ ID NO: 31; CDR2) GDLYYYDSSGYPRYYFDY (SEQ ID NO: 26;QHYSTSPGFT (SEQ ID NO: 32; CDR3) CDR3) F5 SSNYYWG (SEQ ID NO: 36; CDR1)TRSTGSITSNYVH (SEQ ID NO: 42; AIYYSGSTYYNPSLKS (SEQ ID NO: 37; CDR2)CDR1) RIGVAGQWYFDLWGRGTLVTVSS (SEQ ID EDNERPS (SEQ ID NO: 43; CDR2)NO: 38; CDR3) QSYDDSNHISV (SEQ ID NO: 44; CDR3)

TABLE 4Table 4: CDRs (nucleic acid sequences) of the heavy and light chainsof Fabs H9 and F5.Nucleic acid sequences of the CDRs of the Fab antibodies Fab cloneCDRs heavy chain CDRs light chain H9 GCTATGCTATCAGCTG (SEQ ID NO: 27;AGGGCCAGTCAGAGTGTTAGCAGCA CDR1) GCTACTTAGC (SEQ ID NO: 33; CDR1)GGGATCATCCCTATCTTTGGTACAGCAAAC GGTGCATCCAGCAGGGCCACT (SEQTACGCACAGAAGTTCCAGGG (SEQ ID ID NO: 34; CDR2) NO: 28; CDR2)AGCACTATAGCACCTCACCTGGGTTC GGGGATCTGTATTACTATGATAGTAGTGGTACT (SEQ ID NO: 35; CDR3) TATCCGCGATACTACTTTGACTA (SEQ ID NO: 29; CDR3)F5 AGCAGTAATTACTACTGGGGC (SEQ ID ACCCGCAGCACTGGCAGCATTACCA NO: 39; CDR1)GCAACTATGTGCAC (SEQ ID NO: 45; GCTATCTATTATAGTGGGAGCACCTACTAC CDR1)AACCCGTCCCTCAAGAGT (SEQ ID NO: 40; GAGGATAACGAAAGACCCTCT (SEQ CDR2)ID NO: 46; CDR2) CGTATAGGAGTGGCTGGCCAATGGTATTTCCAGTCTTATGATGACAGCAATCATAT GATCTCTGGGGCCGTGGCACCCTGGTCACTTCTGTC (SEQ ID NO: 47; CDR3) CGTCTCAAGC (SEQ ID NO: 41; CDR3)

Production of the Recombinant, Soluble Fab Clones

The isolated Fab clones with specificity toward the HLA-A2/pp65 complex(H9, F5) were produced in a soluble form in E. coli BL21 cells. TheseFabs which are tagged at the CH1 domain with a hexahistidine sequence,were purified from the periplasmic fraction by metal affinitychromatography. SDS-PAGE analysis revealed the level of purification andthe expected molecular size of the Fab antibodies (FIG. 1 b).

These data demonstrate the isolation of recombinant antibodies withpeptide-specific, MHC restricted binding to the CMV-derived T cellepitope pp65₄₉₅₋₅₀₃ (SEQ ID NO:3).

Example 2 Characterization of HLA-A21pp65-Specific TCR-Like RecombinantAntibodies

Experimental Results

HLA-A2/pp65-Specific TCR-Like Recombinant Antibodies Exhibit BindingCharacteristics and Fine Specificity of a TCR-Like Molecule

The specificity of the two recombinant monoclonal Fab antibodies to theMHC-CMV peptide complex was tested by ELISA (FIGS. 1 c and d). Todetermine the correct folding of the bound complexes and their stabilityduring the binding assays, the ability of the complexes to react withthe conformation-specific mAb, w6/32, that recognizes HLA complexes onlywhen folded correctly and when containing peptide was monitored. Asshown in FIGS. 1 c and d, the soluble Fab Abs reacted only with thespecific HLA-A2/pp65 complex but not with other control HLA-A2/peptidecomplexes containing viral epitopes derived from the TAX protein (e.g.,TAX 11-19; SEQ ID NO:14), Gag (e.g., Gag 77-85; SEQ ID NO:9) or Pol(e.g., Pol 476-484; SEQ ID NO:10), or a variety of tumor-associatedepitopes such as telomerase epitopes [e.g., hTERT 540 (SEQ ID NO:6) orhTERT 865 (SEQ ID NO:8)], melanoma gp100 epitopes [e.g., 209 (SEQ IDNO:7) or 280 (SEQ ID NO:4)], XAGE (SEQ ID NO:12), TARP (SEQ ID NO:13)and MART-1-derived epitopes (e.g., MART 26-35; SEQ ID NO:11) (PascoloS., et al., 1997). Thus, these peptide-specific and MHC-restricted Fabantibodies exhibit the binding characteristics and fine specificity of aTCR-like molecule.

HLA-A2/pp65-Specific TCR-Like Recombinant Antibodies Specifically BindMHC-Peptide Complexes Presented on Cells

To demonstrate that the isolated Fab antibodies can bind the specificMHC-peptide complex not only in the recombinant soluble form, but alsoin the native form, as expressed on the cell surface, the presentinventors used murine TAP2 (transporter associated with antigenpresentation)-deficient RMA-S cells transfected with the human HLA-A2gene in a single-chain format (Pascolo S., et al., 1997)(HLA-A2.1/Db-β₂m single chain, RMA-S-HHD cells). The pp65₄₉₅₋₅₀₃ peptideand control peptides were loaded on RMA-S-HHD cells and the ability ofthe selected Fab Abs to bind to peptide-loaded cells was monitored byflow cytometry. Peptide-induced MHC stabilization of the TAP2 mutantRMA-S-HHD cells was demonstrated by the reactivity of mAbs w6/32 (HLAconformation dependent) and BB7.2 (HLA-A2 specific) with peptide-loaded,but not unloaded cells (data not shown). As shown in FIGS. 2 b and d,Fabs H9 and F5 reacted only with pp65-loaded RMA-S-HHD cells, but notwith cells loaded with the EBV derived peptide. Similar results wereobserved in FACS analysis using 10 other HLA-A2-restricted peptides(data not shown).

In addition, the present inventors used the TAP⁺ EBV-transformedB-lymphoblast HLA-A2⁺ JY cells as APCs. These cells have normal TAP;consequently, peptide loading is facilitated by the exchange ofendogenously derived peptides with HLA-A2-restricted peptides suppliedexternally by incubation of the cells with the desired peptides. Asshown in FIGS. 2 a and c, the Fab antibodies recognize only JY cellsloaded with the specific pp65 peptide to which they were selected, butnot with control HLA-A2-restricted peptides derived from melanoma gp100[G9-154 (SEQ ID NO:15) and G9-280 (SEQ ID NO:4) epitopes] and MART1peptides (SEQ ID NO:11), or a telomerase human telomerase reversetranscriptase (hTERT)-derived peptide (T540 epitope; SEQ ID NO:6). As acontrol, peptide-loaded HLA-A2⁻/HLA-A1⁺ APD B cells were used. Nobinding of the Fab Abs to these cells was observed (data not shown).These results demonstrate the ability of the selected Fabs to detectspecifically complexes of HLA-A2 in association with the pp65₄₉₅₋₅₀₃peptide (SEQ ID NO:3), on the surface of cells.

These results demonstrate the fine specificity of the recombinant Fabclones H9 and F5 to soluble or membrane-presented CMV-MHC class Icomplex.

Example 3 Generation of Multivalent Antibody Forms and their Binding toPeptide-Pulsed APCS

Experimental Results

Increased Avidity of Fab Tetramers to Peptide Pulsed APCs

Fab fragments w/o peptide isolated from the phage library aremonovalent. To increase the avidity of these fragments, Fab tetramerswere generated. This approach was previously used to increase thebinding avidity of peptide-MHC complexes to the TCR or to increase thesensitivity of recombinant Ab molecules (Cloutier S M., et al., 2000).To form a Fab tetramer with H9, a BirA tag sequence for site-specificbiotinylation was introduced at the C-terminus of the light chain. TheFab domains were expressed separately in E. coli and were refolded invitro followed by purification and in vitro biotinylation using the E.coli-derived BirA enzyme (Cohen C J., et al., 2002). H9 Fab tetramerswere generated with a fluorescently labeled streptavidin and theirreactivity was examined by flow cytometry with JY pulsed cells. As shownin FIG. 3 a the fluorescence intensity measured on peptide-pulsed JYcells with the H9 Fab tetramer was significantly higher compared to thereactivity of the H9 Fab monomer. The specificity, however, was notaltered (FIG. 3 c).

Increased Avidity of Whole IgG Antibodies to Peptide Pulsed APCs

Another strategy for increasing the avidity was by creating a whole IgGantibody molecule which is bivalent. To transform the recombinant Fabfragment into a whole IgG molecule, eukaryotic shuttle expressionvectors containing the constant regions of IgG1 for the heavy chain anda vector containing the constant domain of a kappa light chain wereused. Recombinant H9 Fab-derived IgG was produced from these expressionvectors by co-transfection of the two constructs into human embryonickidney HEK293 cells. After proper selection and generation of stablesecreting clones, purified TCR-like whole IgG molecules were producedand tested for binding specifically towards APCs pulsed with thepp65495-503 peptide. As shown in FIG. 3 b, the binding specificity ofthe whole IgG molecule was maintained. As expected, the fluorescenceintensity observed with the IgG was significantly higher compared tothat of the Fab monomer. JY cells pulsed with control peptide (derivedfrom gp100) were incubated with the three H9 constructs (monomer,tetramer, whole IgG Ab) to confirm specificity (FIG. 3 c).

These results demonstrate the generation of bivalent (IgG) or tetramericFab antibodies and the increased avidity, yet without compromisingspecificity of the recombinant antibodies to the CMV-MHC class Icomplex.

Example 4 The TCR-Like Antibodies of the Invention are Highly Specificand Sensitive to MHC-CMV Peptide Complexes

Experimental Results

Determination of Binding Affinity of the Recombinant TCR-Like Antibodies

Binding affinity determination of the H9 Ab was performed by surfaceplasmon resonance (SPR) analysis using streptavidin sensor chips coatedwith biotinylated HLA-A2/pp65 or control HLA-A2/EBV complexes. Theapparent affinity of the monomeric/IgG forms of the H9 Ab indicatedK_(D) values of 8 nM and 5 nM, respectively. The time necessary forbinding of the H9 Fab/IgG Ab to the specific complexes (K_(on)) was1.05×10⁵ 1/Ms and 5.99×10⁵ 1/Ms, respectively. The dissociation rate (Kdor K_(off)) of the H9 Fab was 8.79×10⁴ 1/s compared to the H9 IgG Ab,which was 3.52×10⁻³ 1/s (FIGS. 4 a, b). No significant binding of theantibodies was detected when control HLA-A2/EBV complexes wereimmobilized to the sensor chip (FIG. 4 c).

The Recombinant TCR-Like Antibodies are Highly Specific to the MHC-pp65Complex

To study the sensitivity of ligand recognition by the Fab and itsderivatives the reactivity threshold was examined by peptide titrationon JY cells which were pulsed with different concentrations of the pp65495-503 peptide. As shown in FIGS. 5 a and b, peptide titration ofpulsed JY demonstrated that the staining intensity was dependent on theconcentration of the peptide used for pulsing, and that peptideconcentrations at the low nM range were sufficient for Fab tetramer(FIG. 5 b) but not for the monomer (FIG. 5 a). Thus, the tetrameric formof H9 Fab was able to detect much lower numbers of peptide/HLA-A2complexes on the surface of peptide-pulsed JY cells than the monomer.Similar results were observed with the whole IgG molecule (data notshown). Overall, these and additional studies revealed that the H9tetramer and IgG molecules are capable of detecting HLA-A2/pp65complexes on cells pulsed with as low as ˜100 nM pp65₄₉₅₋₅₀₃ peptide.

The Recombinant TCR-Like Antibodies can Detect Low Amounts of MHC-pp65Complexes Presented on Cells in a Mixed Population of Cells

The TCR-like Fab were further used to detect APCs bearing the specificpeptide-MHC complexes in a heterogeneous cell population. This canverify the ability of the TCR-like Fab molecules to detect complexes onindividual cell samples in a mixed cell population. To simulate thesituation of a heterogeneous population of cells in which only a smallfraction might express the specific peptide-MHC complex, pp65 peptidepulsed JY cells were mixed with HLA-A2⁻/HLA-A1⁺ APD B cells at variousratios and the reactivity of H9 Fab was analyzed by flow cytometry. Asshown in FIG. 5 c, staining with H9 Fab tetramer allows accurateidentification of the admixed pp65 JY pulsed cells that express on theirsurface HLA-A2/pp65 complexes, using a simple one-color flow cytometryanalysis. Using various ratios of mixtures between pulsed and nonpulsedcells, the H9 Fab was shown capable of detecting as low as 5% pp65 JYpulsed cells within a background population of 95% nonpulsed cells (FIG.5 c).

Altogether, these results demonstrate detection of cell subpopulationbearing CMV peptide-MHC complexes.

Example 5 The TCR-Like Antibodies of the Invention can DetectHLA-A2/pp65 Complexes on Surface of Viral-Infected Cells

Experimental Results

Detection of HLA-A2/pp65 Complexes on the Surface of Virus-InfectedCells

To test the ability of the isolated Fab to bind specifically HLA-A2/pp65complexes produced under naturally occurring physiological Antigen (Ag)processing, HLA-A2 positive fibroblasts were infected with the CMVlaboratory strain AD169 at multiplicity of infection (MOI) of 0.5 (FIGS.6 a-1). HLA-A2 negative fibroblasts infected with the virus, were usedas control in addition to uninfected HLA-A2 negative and positive cells.72 hours after infection, infected and control cells were incubated withthe tetrameric form of H9. To verify the expression of HLA-A2 moleculeson the surface of infected, versus uninfected cells, the humanfibroblasts were also stained with PE-labeled BB7.2. Confirmation forefficiency of virus infection was monitored with anti pp65 mAb and thesecondary antibody FITC-labeled anti mouse IgG. As shown in FIGS. 6 aand c, there was a somewhat decrease in the expression of HLA-A2complexes on the surface of the virus infected cells, due to the viruswell known down regulation mechanism of the MHC expression. However,despite the relatively low amount of HLA-A2 expressed on the cellsurface, there was still specific staining of infected cells with the H9tetramer (FIGS. 6 e and g), suggesting that the isolated antibody wasable to detect not only complexes presented on peptide pulsed APCs butalso specific MHC-peptide complexes expressed after active and naturallyoccurring endogenous intracellular processing. The H9 Ab showed nobinding at all in the control uninfected cells (FIGS. 6 g and h) as wellas in the HLA-A2 negative cells (FIG. 6 f), indicating its finespecificity towards HLA-A2/pp65 complexes presented on the cell surface.Staining with the anti pp65 mAb revealed the expression of the pp65protein after successful infection of the fibroblasts (FIGS. 6 i and j).

The specificity of the H9 Ab was verified using a control TCR-like Ab(2F1) which recognizes specifically class I MHC complexes in associationwith the gp100 280-288 peptide. No staining was visible in this assay,confirming again the H9 tetramer's specificity (data not shown).

These results demonstrate, for the first time, the ability to follow theCMV-MHC class I complexes on the cells surface of APC as well as insideinfected cells.

Example 6 The TCR-Like Antibodies of the Invention can Compete with CTLSon Specific HLA-A2/pp65 Sites and Thereby Prevent CTL-MediatedCytotoxicity

Experimental Results

The H9 Ab can Prevent CTL-Mediated Cytotoxicity Directed Against theHLA-A2-pp65 Complex

The specificity of the H9 Ab to the MHC-pp65 495-503 complex presentedon cells was further demonstrated by the specific inhibition ofCTL-mediated cell killing by the H9 antibody. Briefly, fibroblast cellswere radioactively labeled with S³⁵⁻methionine before infection with theCMV virus and 72 hours later the cells were incubated with H9 Ab. CTLsfrom a line targeted to the pp65 (495-503) epitope were added at atarget (i.e., fibroblast cells)—effector (i.e., CTL) ratio of 1:10 andincubated for five hours. Cells incubated with anti-HLA-A2 W6/32 MAbwere used as positive control, while cells without any Ab incubationserved as a reference for maximal killing. As shown in FIG. 6 m, maximalpercentage of killing was observed in the virus infected cells whichwere not incubated with Abs (CMV CTL alone). However, incubation withthe H9 IgG Ab exhibited ˜60% blockage of killing by the CTLs (CMVCTL+H9).

The cytotoxicity assay demonstrated the capability of the isolatedantibody to recognize specifically complexes presented on virus infectedcells and its potential to compete with the same sites recognized byCTLs, leading to the blockage of killing by these effector cells.

Example 7 The TCR-Like Antibodies of the Invention are Valuable Toolsfor Following the Dynamics of HLA-A2/pp65 Expression in Cells Infectedwith the CMV Virus

Experimental Results

The Dynamics of HLA-A2/pp65 Complex Expression in Cells Infected withWild-Type and Mutant Virus

The fact that the H9 Ab was able to detect specific complexes on virusinfected cells enabled to follow the expression levels of the complexesthroughout the virus infection cycle. Based on precedent results whichshowed down regulation of MHC class I expression after viral infection(Ahn, K. et al. 1996), the present inventors investigated whether thegeneration and presentation of HLA-A2/pp65 complexes throughout varioustime points after infection is influenced by the down regulationmechanism. To this end two strategies were employed; (i) theintracellular versus extracellular staining with H9 or anti-HLA-A2 BB7.2Abs which enabled to determine if the level of the complexesgeneration/expression is correlated with their uptake to the cellsurface; (ii) the usage of a mutant strain of CMV which does not inducedown regulation of MHC class I. The level of expression of HLA-A2/pp65complexes in cells infected with the wild type AD169 strain was comparedto that in cells infected with the mutant strain. For this purpose, thegenetically modified CMV strain RV798 (Jones T R and Sun L., 1997),which lacks most of the genes responsible for the down regulationmechanism of MHC class I (US2 to US11 genes), was employed.

As shown in FIGS. 7 a-t, 8 a-t and 9 a-y, the general expression ofHLA-A2 class I MHC was followed throughout four time points (36, 72, 96and 120 hours) after cell infection with AD169 WT CMV strain (FIGS. 7a-t) and RV798 mutant CMV strain (FIGS. 8 a-t), as well as theexpression of specific HLA-A2 complexes in association with the pp65495-503 peptide using the H9 IgG Ab. The infection efficiency wasmonitored by following the expression of the pp65 protein in infectedcells through the use of an anti-pp 65 MAb. Detection with H9 or BB7.2Abs was performed in each time point by intracellular and extracellularstaining. To verify the specificity of the reagents used for detection,especially the reactivity of the anti-HLA-A2/pp65 495-503 TCR-likeantibody, controls which were uninfected HLA-A2 positive fibroblasts(FIGS. 9 a-t) or CMV infected human fibroblasts that are HLA-A2 negative(FIGS. 9 u-y) were used. The results show progressive expression of pp65in cells infected with wild-type (FIGS. 7 e, j, o, t) and mutant (FIGS.8 e, j, o, t) CMV strains while in non-infected cells (FIGS. 9 e, j, o,t) no expression was observed. The expression of pp65 in cells that wereinfected with the mutant stain RV798 was somewhat higher. Staining withthe anti pp65 Ab also indicated that the cells begin to express the pp65protein less than 36 hours after infection (data not shown). These dataare in agreement with previous studies (Soderberg-Naucler C., et al.,1998). Expression of HLA-A2 on the surface of cells infected withwild-type virus clearly showed a phenotype involving significant downregulation of HLA-A2 expression (FIGS. 7 c, h, m and r) compared to theuninfected fibroblasts (FIGS. 9 c, h, m, r). This down regulation isincreased over time through the progression of the time points. Also,the intracellular expression of HLA-A2 in infected cells seemed to behigher than the amount in the uninfected cells (Compare FIGS. 7 d, i, nand s to FIGS. 9 d, i, n and s, respectively). These data are inagreement with previous studies (Ahn K., et al., 1996).

When cells were infected with wild-type virus, a specific and gradualincrease in staining with the H9 IgG TCR-like antibody was observedindicating the generation of HLA-A2/pp65 495-503 complexes insideinfected cells (FIGS. 7 b, g, l and q) as well as their presentation onthe cell surface (FIGS. 7 a, f, k and p). However, although the amountof complexes which bear the pp65 495-503 peptide seemed to be quite lowat the cell surface (e.g., compare FIG. 7 f with 7 g), intracellularstaining of these specific complexes revealed a very significant largepool of complexes inside the cell. This might indicate that although thepp65 is well processed inside the cell and its peptides are deposited onthe class I MHC, it is avoided from being displayed on the cell surfaceas part of the virus evasion mechanisms. Interestingly, there was nocorrelation between HLA-A2 down regulation as clearly observed throughthe progression of time and the significant increase in theintracellular pools of HLA-A2/pp65 495-503 complexes or their expressionon the cell surface. Most striking is that after 120 hours theexpression of HLA-A2 is very low however both intracellular pools arevery high and surface expression is significant.

The Reactivity of the H9 IgG Molecule to the MHC-CMV pp65 PeptideComplex Both Inside and on the Surface of Cells is Highly Specific

Non-infected HLA-A2 positive cells were stained with anti-HLA-A2antibody BB7.2 both inside (FIGS. 9 d, i, n, s) and on the surface(FIGS. 9 c, h, m, r). As shown, there were no observed alterations inHLA-A2 expression inside the cells as well as its presentation on thecell surface throughout the time points tested after infection. Incontrary to the infected fibroblasts, the amount of complexes asdetermined using the BB7.2 antibody on the cell surface of non-infectedcells seemed to be higher than their amount inside the cells (compareFIGS. 9 c, h, m, r with FIGS. 9 d, i, n, s, respectively). No pools ofcomplexes were observed inside the cells (FIGS. 9 d, i, n, s) as seen inthe infected fibroblasts (FIGS. 7 d, i, n, s). This implies that theHLA-A2 complexes expressed inside the uninfected cells are freelypresented on the cell surface, in contrast to the infected cells (seeFIGS. 7 d, i, n, s). In contrast, the H9 TCR-like antibody was notreactive with uninfected cells both inside (FIGS. 9 b, g, l, q) and onthe cell surface (FIGS. 9 a, f, k, p), indicating its fine specificitytowards its antigen.

When HLA-A2 negative human fibroblasts were infected with wild-type CMV,pp65 expression was clearly observed (FIG. 9 y), however, no reactivitywith the anti-HLA-A2 antibody (FIGS. 9 w, x) or the H9 TCR-like antibody(FIGS. 9 u, v) was observed inside or on the surface of the infectedcells indicating the highly specific reactivity of the molecules.

The presentation of HLA-A2/pp65 complexes was further examined bothinside the cells and on their surface less than 24 hours afterinfection. These studies demonstrated that although pp65 is expressed,there is no presentation of its peptides on HLA-A2 molecules (Data notshown).

FIGS. 8 a-t follow the dynamics of antigen presentation in the mutantstrain RV798. The infected cells were efficiently infected with thevirus as observed from the staining with anti-pp65 (FIGS. 8 e, j, o, t).It was clearly observed that the effect of the mutant virus on HLA-A2expression inside and on the surface of infected cells was diminished,thus the mutant virus no longer significantly down regulates HLA-A2expression, as expected. When using the H9 IgG TCR-like antibody,similar to the results observed with wild-type CMV, a gradual increaseover time of intracellular pools of HLA-A2/pp65 495-503 complexes insideinfected cells was observed (FIGS. 8 b, g, l, q) as well as theirgradual appearance on the cell surface (FIGS. 8 a, f, k, p). Also, itwas quite evident that the number of HLA-A2/pp65 495-503 complexesinside the infected cells was higher than those on the cell surface(Compare FIGS. 8 b, g, l, q to FIGS. 8 a, f, k, p, respectively). Thismay indicate that although the mutant virus does not activate the downregulation mechanism, there are still HLA-A2 pools as well as specificHLA-A2/pp65 pools inside the cells, which are avoided from beingpresented on the cell surface.

In general, these results present the usage of the H9 Ab to follow thedynamic expression and kinetics of HLA-A2/pp65 495-503 presentationintracellularly and on the surface of infected cells as a function oftime after viral infection. Most striking is the observation that thereis no correlation between class I MHC down regulation induced bywild-type virus and the generation/presentation of the viral specificHLA-A2/pp65 495-503 complex. On the contrary, the down regulation didnot affect the generation of a significant and large intracellular poolof viral complexes and their appearance over time on the cell surface.Similar studies using the H9 antibody and a mutant virus that abolishesclass I MHC down regulation showed a similar pattern of expressioninside the cell and on its surface with somewhat increased number ofcomplexes on both compared to wild-type virus especially between 24-72hours after infection.

Example 8 The TCR-Like Antibodies of the Invention can be Used toQuantify the Number of HLA-A2/pp65 Complexes on Viral Infected Cells

The knowledge of the number of complexes presented on the cell surfacecan be used to understand how the immune system identifies viralinfection. Related to the studies presented herein, the presentinventors attempted to quantify and compare the number of complexesgenerated inside the infected cells to those presented on the cellsurface, as follows.

Experimental Results

Quantization of the Number of HLA-A2/pp65 Complexes on the Surface ofInfected Cells

The unique H9 IgG TCR-like antibody enables the present inventors todirectly quantify the number and percentage of specific HLA-A2/pp65complexes among HLA-A2-derived complexes which are displayed on the cellsurface. Staining of virus infected cells with the H9 IgG TCR-likeantibody enabled the present inventors to directly count the number ofcomplexes on the surface of the infected cells using a PE-labeled antikappa secondary monoclonal antibody that generates a 1:1 bindingstoichiometry with the H9 IgG molecule. The level of fluorescenceintensity resulting from specific reactivity of the H9 IgG antibody oninfected cells can be directly correlated with the fluorescenceintensities of calibration beads with known numbers PE molecules perbead (QuantiBRITE PE beads; BD Biosciences), using simple flow cytometrycalibrations. This strategy enabled the present inventors to determinethe number of PE molecules bound to the cells and thereby the number ofsites which are bound by the H9 antibody.

In agreement to the results presented on FIGS. 7-9 (Example 7,hereinabove), there was an immediate and massive down regulation ofHLA-A2 complexes (using the BB7 Ab) from the cell surface afterinfection with the CMV wild-type strain (FIG. 10 d). In all time pointsthere were about 5,000 complexes observed on the cell surface comparedto ˜25,000 complexes in the uninfected cells, implying that there wasover 85% decrease in the amount of HLA-A2 complexes presented on thecell surface (FIG. 10 d). The number of HLA-A2 complexes inside thecells in infected vs uninfected cells remained almost the same (FIG. 10c). The number of HLA-A2/pp65 complexes presented on the cell surfacewas gradually increased over time (FIG. 10 b). Specific complexes wereobserved using the H9 antibody starting at 36 hours after infection andthe number reached to approximately 400 sites/cell 120 hours afterinfection (FIG. 10 c). This implies that 120 hours after infection withthe virus, about 10%-15% of the HLA-A2 complexes presented on the cellsurfaces bear the pp65 495-503 peptide. Interestingly, the number ofthese specific complexes inside the cells reaches to ˜2000/cell after120 hours (FIG. 10A). This number is close to the total number of HLA-A2complexes inside the cell, suggesting that most of the HLA-A2 complexeswhich accumulate inside infected cells are HLA-A2/pp65. This might alsosuggest that most of these specific complexes which are generated insidethe cells are avoided from being presented on the surface.

Using the mutant virus, the same number of HLA-A2 molecules on the cellsurface was observed as in the uninfected cells (FIG. 10 d). The numberof sites reached to approximately 20,000 (FIG. 10 d). However, thenumber of complexes quantified inside the cells was significantly higherthan the number observed in the uninfected cells, and approached to˜10,000 (FIG. 10 c) compared to ˜1,000 in the uninfected cells (FIG. 10c). As for HLA-A2/pp65 complexes, there were ˜400 sites detected on thecell surface (FIG. 10 b), implying that similar to cells infected withwild-type virus most of viral HLA-A2/pp65 complexes are avoided frombeing transported to the cell surface. The percentage of these complexesamongst HLA-A2 complexes on the cell surface is very low. However, thenumber of HLA-A2/pp65 complexes inside the infected cells reached toapproximately 3,000 (FIG. 10 a) in each time point after 72 hours thusuntil 120 hours after infection there is an accumulation of the specificcomplexes inside the cell. This accumulation might lead to theobservation that after this time point, most of the complexes inside thecell are composed of HLA-A2/pp65.

These data provide a quantitative measure to the observation thatspecific HLA-A2/pp65 complexes are being generated in large amounts andaccumulated inside the infected cell in a mechanism that is independentto the overall down regulation of HLA-A2 molecules in these cells. Theaccumulation was observed with wild-type and mutant virus strains andfor both the accumulated HLA-A2/pp65 complexes were avoided from beingpresented in large amounts on the cell surface.

These results visualize large intracellular pools of the viral complexesafter infection, follow and quantify their expression on the surface.These results demonstrate that despite significant down regulation ofMHC expression by wild-type virus large pools of specific viralcomplexes are generated intracellularly, and their export to the cellsurface occurs in a limited quantity. These studies describe the firstattempt to directly visualize and analyze the dynamics of a naturallyoccurring viral-derived human MHC-peptide complex after viral infection.

The data also demonstrate the ability of the TCR-like antibody of theinstant application to detect and accurately quantify the number ofHLA-A2/peptide complexes on the surface of infected cells undernaturally occurring intracellular processing. These results can be usedto follow the effectiveness of viral strategies for immunization.

Example 9 Visualization Through Confocal Microscopy Imaging ofHLA-A2/pp65 Expression in Virus-Infected Cells

Experimental Results

Visualization Through Confocal Microscopy Imaging of HLA-A2/pp65Expression in Virus-Infected Cells

Confocal microscopy of CMV infected cells stained with the H9 IgGTCR-like antibody enabled the present inventors to visualize and imagethe specific HLA-A2/pp65 complexes generated inside the cells, as wellas their display on the cell surface. Moreover, it enabled the presentinventors to localize the complexes inside the cell during the virusinfection cycle.

CMV infected cells were harvested every 24 hours for 5 days. At eachtime point cells were stained with the H9 Ab, and anti human alexafluor⁴⁸⁸ as a secondary Ab. The cells were also stained intracellularlywith the H9 Ab, anti calnexin, cis Golgi matrix protein (GM130), andanti pp65 Ab, after fixation and permeabilization. Secondary antibodyfor the ER marker, Golgi marker and anti pp65 was anti mouse alexafluor⁵⁹⁴. Noninfected fibroblast cells were used as a control.

The results of these assays further demonstrate and image thesignificant pool of specific HLA-A2/pp65 complexes generated insideinfected cells (FIGS. 11 a-o, 12 a-o). The data also show that thespecific complexes are densely colocalized with the cis-golgi apparatus(FIGS. 11 a-o). This co-localization is observed clearly after 24 hoursin comparison with the later time points, in which the complexes aremore widely distributed and co-localized to the ER/cytosol as indicatedby co-staining with the various localization markers (FIGS. 12 a-o).Additionally, as time progresses, a significant enlargement of the Golgiapparatus is observed, as part of the morphological changes of theinfected cells. Extracellular staining of the HLA-A2/pp65 complexesshowed their display on the cell surface only after 72 hours postinfection (FIGS. 13 a-e). These results are with complete agreement withthe flow cytometry analysis of the kinetic of HLA-A2/pp65 epitopepresentation as shown in FIGS. 7 a-t, 8 a-t and 9 a-y. Confocalmicroscopy analysis of control noninfected cells showed no staining withthe H9 Ab (FIGS. 13 f-h), indicating its fine specificity towards theHLA-A2/pp65 complexes. Staining with anti pp65 Ab confirmed theeffectiveness of the viral infection in the experiments (FIGS. 13 i-j).

These results visualize the present inventors' finding that specificHLA-A2/pp65 complexes are being generated and accumulate in infectedcells and are localized in the Golgi compartment. They are preventedfrom being displayed on the cell surface at early time points and only72 hours after infection they can be imaged on the cell surface. Thefact that the specific complexes are prevented from being displayed onthe cell surface is only temporary. Progressed time scales showed thatthe complexes are being significantly displayed on the cell surface. Theintermediate time points clearly show that the complexes are lessco-localized with the Golgi due to their movement to the cell membrane.The phenomena of Golgi enlargement is usually attributed to an extensivesynthesis of proteins after viral infection. These results can implythat this enlargement is also due to the specific accumulation ofcomplexes in the Golgi.

Example 10 CMV pp64 MHC Restricted Peptides

Tables 5-70 hereinbelow provide the user parameters and scoringinformation used to select CMV PP64 restricted peptides (each of 9 or 10amino acids in length) of various HLA molecules. The analysis wasperformed using the Bimas software[hypertexttransferprotocol://worldwideweb-bimas (dot) cit (dot) nih(dot) gov/molbio/hla_bind/]. The scoring results and the sequences ofthe selected peptides (according to each user parameters and scoringinformation) are provided in Table 137 in Example 11, hereinbelow. TheCMV PP64 kDa protein used for analysis is provided by SEQ ID NO:52[(GenBank Accession No. P18139; PP65_HCMVT 64 kDa lower matrixphosphoprotein—Human cytomegalovirus (strain Towne) (HHV-5) (Humanherpesvirus 5)].

TABLE 5 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A1 length selected forsubsequences to be scored  9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported  15 back in scoring output table

TABLE 6 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A1 length selected forsubsequences to be scored  10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported  20 back in scoring output table

TABLE 7 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A_0201 length selected forsubsequences to be scored  9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported  48 back in scoring output table

TABLE 8 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A_0201 length selected forsubsequences to be scored  10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported  53 back in scoring output table

TABLE 9 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A_0205 length selected forsubsequences to be scored  9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported  50 back in scoring output table

TABLE 10 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A_0205 length selected forsubsequences to be scored  10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported  47 back in scoring output table

TABLE 11 method selected to limit number of results cutoff score cutoffscore selected   1 HLA molecule type selected A24 length selected forsubsequences to be scored   9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported  61 back in scoring output table

TABLE 12 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A24 length selected forsubsequences to be scored  10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported  76 back in scoring output table

TABLE 13 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A3 length selected forsubsequences to be scored  9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported  23 back in scoring output table

TABLE 14 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A3 length selected forsubsequences to be scored  10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported  21 back in scoring output table

TABLE 15 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A68.1 length selected forsubsequences to be scored  9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported  79 back in scoring output table

TABLE 16 method selected to limit number of results cutoff score cutoffscore selected  1 HLA molecule type selected A68.1 length selected forsubsequences to be scored  10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported  77 back in scoring output table

TABLE 17 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_1101 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 5 in scoring output table

TABLE 18 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_1101 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back 4 in scoring output table

TABLE 19 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3101 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 10 in scoring output table

TABLE 20 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3101 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back 17 in scoring output table

TABLE 21 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3302 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 39 in scoring output table

TABLE 22 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3302 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back 34 in scoring output table

TABLE 23 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B14 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 61 in scoring output table

TABLE 24 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B14 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back 60 in scoring output table

TABLE 25 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B40 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 36 in scoring output table

TABLE 26 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B40 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back 42 in scoring output table

TABLE 27 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B60 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 41 in scoring output table

TABLE 28 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B60 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back 49 in scoring output table

TABLE 29 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B61 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 44 in scoring output table

TABLE 30 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B61 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back 45 in scoring output table

TABLE 31 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B62 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back 57 in scoring output table

TABLE 32 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B62 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 54

TABLE 33 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B7 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back in scoring output table 54

TABLE 34 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B7 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 62

TABLE 35 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B8 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back in scoring output table 12

TABLE 36 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B8 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 14

TABLE 37 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2702 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back in scoring output table 71

TABLE 38 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2702 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 72

TABLE 39 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2705 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back in scoring output table 276

TABLE 40 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2705 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 283

TABLE 41 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3501 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back in scoring output table 72

TABLE 42 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3501 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 81

TABLE 43 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3701 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back in scoring output table 90

TABLE 44 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3701 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 100

TABLE 45 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3801 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported back in scoring output table 50

TABLE 46 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3801 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported back in scoring output table 59

TABLE 47 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3901 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 100 back in scoring output table

TABLE 48 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3901 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 102 back in scoring output table

TABLE 49 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3902 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 61 back in scoring output table

TABLE 50 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3902 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 69 back in scoring output table

TABLE 51 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_4403 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 47 back in scoring output table

TABLE 52 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_4403 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 56 back in scoring output table

TABLE 53 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5101 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 139 back in scoring output table

TABLE 54 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5101 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 127 back in scoring output table

TABLE 55 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5102 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 149 back in scoring output table

TABLE 56 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5102 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 140 back in scoring output table

TABLE 57 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5103 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 89 back in scoring output table

TABLE 58 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5103 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 86 back in scoring output table

TABLE 59 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5201 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 111 back in scoring output table

TABLE 60 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5201 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 120 back in scoring output table

TABLE 61 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5801 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 55 back in scoring output table

TABLE 62 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5801 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 50 back in scoring output table

TABLE 63 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0301 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 99 back in scoring output table

TABLE 64 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0301 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 91 back in scoring output table

TABLE 65 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0401 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 88 back in scoring output table

TABLE 66 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0401 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 96 back in scoring output table

TABLE 67 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0602 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 115 back in scoring output table

TABLE 68 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0602 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 117 back in scoring output table

TABLE 69 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0702 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 543 number of top-scoringsubsequences reported 61 back in scoring output table

TABLE 70 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw _0702 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence551 number of subsequence scores calculated 542 number of top-scoringsubsequences reported 73 back in scoring output table

Example 11 CMV PP65 MHC Restricted Peptides

Tables 71-136 hereinbelow provide the user parameters and scoringinformation used to select CMV PP65 restricted peptides (each of 9 or 10amino acids in length) of various HLA molecules. The analysis wasperformed using the Bimas software[hypertexttransferprotocol://worldwideweb-bimas (dot) cit (dot) nih(dot) gov/molbio/hla_bind/]. The scoring results and the sequences ofthe selected peptides (according to each user parameters and scoringinformation) are provided in Table 137, hereinbelow. The CMV PP65 kDaprotein used for analysis is provided by SEQ ID NO:53 [GenBank AccessionNo. P06725; PP65_HCMVA 65 kDa lower matrix phosphoprotein—Humancytomegalovirus (strain AD169) (HHV-5) (Human herpesvirus 5)].

TABLE 71 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A1 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 15 back in scoring output table

TABLE 72 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A1 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 20 back in scoring output table

TABLE 73 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_0201 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 48 back in scoring output table

TABLE 74 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_0201 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 53 back in scoring output table

TABLE 75 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_0205 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 51 back in scoring output table

TABLE 76 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_0205 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 47 back in scoring output table

TABLE 77 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A24 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 64

TABLE 78 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A24 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back in scoring output table 76

TABLE 79 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A3 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 23

TABLE 80 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A3 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back in scoring output table 21

TABLE 81 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A68.1 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 79

TABLE 82 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A68.1 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back in scoring output table 77

TABLE 83 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_1101 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 5

TABLE 84 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_1101 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back in scoring output table 4

TABLE 85 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3101 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 10

TABLE 86 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3101 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back in scoring output table 17

TABLE 87 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3302 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 40

TABLE 88 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected A_3302 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back in scoring output table 36

TABLE 89 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B14 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 65

TABLE 90 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B14 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back in scoring output table 63

TABLE 91 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B40 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in scoring output table 35

TABLE 92 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B40 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 42 back in scoring output table

TABLE 93 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B60 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 42 back in scoring output table

TABLE 94 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B60 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 50 back in scoring output table

TABLE 95 User Parameters and Scoring Information User Parameters andScoring Information method selected to limit number of results cutoffscore cutoff score selected 1 HLA molecule type selected B61 lengthselected for subsequences to be scored 9 echoing mode selected for inputsequence Y echoing format numbered lines length of user's input peptidesequence 561 number of subsequence scores calculated 553 number oftop-scoring subsequences reported 44 back in scoring output table

TABLE 96 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B61 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 46 back in scoring output table

TABLE 97 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B62 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 57 back in scoring output table

TABLE 98 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B62 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 54 back in scoring output table

TABLE 99 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B7 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 57 back in scoring output table

TABLE 100 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B7 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 63 back in scoring output table

TABLE 101 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B8 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 12 back in scoring output table

TABLE 102 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B8 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 15 back in scoring output table

TABLE 103 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2702 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 74 back in scoring output table

TABLE 104 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2702 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 74 back in scoring output table

TABLE 105 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2705 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 282 back in scoring output table

TABLE 106 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_2705 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 287 back in scoring output table

TABLE 107 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3501 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back 74 in scoring output table

TABLE 108 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3501 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back 82 in scoring output table

TABLE 109 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3701 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back in 93 scoring output table

TABLE 110 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3701 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back 103 in scoring output table

TABLE 111 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3801 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back 51 in scoring output table

TABLE 112 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3801 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back 59 in scoring output table

TABLE 113 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3901 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back 104 in scoring output table

TABLE 114 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3901 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back 105 in scoring output table

TABLE 115 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3902 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back 63 in scoring output table

TABLE 116 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_3902 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back 70 in scoring output table

TABLE 117 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_4403 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back 47 in scoring output table

TABLE 118 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_4403 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported back 57 in scoring output table

TABLE 119 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5101 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back 141 in scoring output table

TABLE 120 method selected to limit number of results cutoff score 1 HLAmolecule type selected B_5101 length selected for subsequences to bescored 10 echoing mode selected for input sequence Y echoing formatnumbered lines length of user’s input peptide sequence 561 number ofsubsequence scores calculated 552 number of top-scoring subsequencesreported back 128 in scoring output table

TABLE 121 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5102 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user’s input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported back 151 in scoring output table

TABLE 122 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5102 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 140 back in scoring output table

TABLE 123 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5103 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 90 back in scoring output table

TABLE 124 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5103 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 86 back in scoring output table

TABLE 125 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5201 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 114 back in scoring output table

TABLE 126 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5201 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 120 back in scoring output table

TABLE 127 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5801 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 55 back in scoring output table

TABLE 128 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected B_5801 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 49 back in scoring output table

TABLE129 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0301 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 103 back in scoring output table

TABLE 130 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0301 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 93 back in scoring output table

TABLE 131 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0401 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 90 back in scoring output table

TABLE 132 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0401 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 98 back in scoring output table

TABLE 133 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0602 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 119 back in scoring output table

TABLE 134 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0602 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 120 back in scoring output table

TABLE 135 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0702 length selected forsubsequences to be scored 9 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 553 number of top-scoringsubsequences reported 62 back in scoring output table

TABLE 136 method selected to limit number of results cutoff score cutoffscore selected 1 HLA molecule type selected Cw_0702 length selected forsubsequences to be scored 10 echoing mode selected for input sequence Yechoing format numbered lines length of user's input peptide sequence561 number of subsequence scores calculated 552 number of top-scoringsubsequences reported 74 back in scoring output table

Table 137 hereinbelow, depicts subsequence residue listing (Sequence),SEQ ID NO: and scoring results [Rank and Score (the estimate of halftime of disassociation of a molecule containing this subsequence)]obtained according to the user parameters and scoring informationsummarized in Tables 5-136, hereinabove, for HLA restricted peptidesderived from the CMV pp65 (SEQ ID NO:53) or pp64 (SEQ ID NO:52)polypeptides. For each row, a reference to the relevant “user parametersand scoring information Table” is made by indicating the “Table No.” onthe last column.

Lengthy table referenced here US20130101594A1-20130425-T00001 Pleaserefer to the end of the specification for access instructions.

Example 12 Detection of HLA-A2/pp65 Complexes on the Surface ofVirus-Infected Cells of Patients

The ability of the H9 Ab to detect HLA-A2/pp65 complexes was furtherevaluated in heterogeneous population of cells taken from CMV infectedindividuals. Briefly, samples were taken from bone marrow transplanted(BMT) patients whom are under reactivation of CMV infection due toimmuno-suppression. Healthy donors were used as a control to verify theH9 Fab specificity.

Experimental Results

Peripheral blood mononuclear cells (PBMCs) were isolated from samplestaken from BMT patients and healthy donors. The isolated cells werestained with the H9 Ab and the secondary anti human alexa fluor⁴⁸⁸ Ab.For intracellular staining with the H9 Ab, the cells were permeabilizedas described under “General Materials and Experimental Methods”.

Both healthy donors and BMT patients were HLA-A2+ (i.e., express theHLA-A2 allele) as detected by the anti HLA-A2 Ab (BB7.2) and anti mousealexa fluor⁴⁸⁸ Abs (FIG. 16 a and data not shown). Extracellularstaining with the H9 Ab did not detect complexes of the HLA-A2/pp65 onthe surface of infected cells taken from BMT patients or healthycontrols (FIG. 16 b and data not shown). However, as shown in FIGS. 16 cand d, intracellular staining with the H9 Ab demonstrated a significantbinding of the antibody to the infected cells from BMT patients (FIG. 16c) as compared to the control cells taken from healthy donors (FIG. 16d). These results confirm the ability of the isolated H9 Ab to detectspecific HLA-A2/pp65 complexes not only after directed infection withlaboratory strain of the CMV, but also complexes derived from cellsundergoing reactivation of the virus e.g., due to immuno-suppression.

Example 13 Proteasome Inhibitor Effect on HLA-A2/pp65 Complexes in VirusInfected Cells

Experimental Results

The Release of Complexes Accumulation from their Intracellular Locationto the Cell Surface by Proteasome Inhibitor

The proteasome inhibitor acetyl-leucyl-leucyl-norleucinal (ALLN;available from CALBIOCHEM Cat. No. 208750) was used in order tounderstand the mechanism by which complexes are prevented from reachingthe cell membrane. The effect of the proteasome inhibitor was examinedby FACS analysis, while treating the infected cells with ALLN at threetime scales after infection. At each time scale, the cells wereextracellularly stained with the H9 Ab and anti human alexa-flour⁴⁸⁸ asa secondary Ab. As shown in FIGS. 17 a-i there was a significant effectof the inhibitor on the presentation of the complexes on the cellsurface. Presence of the inhibitor at each time scale caused anincreased presentation of the complexes on the cell surface compared tountreated cells. The effect of the increased presentation was moresignificant at the lower time scales, and seamed to reach a steady stateat 96 hours post infection. Control uninfected cells showed no stainingwith the H9 Ab. Thus, incubation with the proteasome inhibitor ALLNincreased presentation of the MHC/pp65 complexes on the cell surface.

SUMMARY

In this study, the present inventors have demonstrated the selection ofrecombinant Fab Abs directed against a human viral T cell epitopederived from CMV, from a large nonimmune human Ab phage library. TheseAbs exhibit an exquisite, very specific, and special binding pattern:they can bind in a peptide-specific manner only to HLA-A2/pp65complexes; hence, these are recombinant Abs with T cell Ag receptor-likespecificity. In contrast to the inherent low affinity of TCRs, thesemolecules display the high affinity binding characteristics of Abs, inthe nM range, while retaining TCR specificity. The present inventorshave demonstrated here the ability of these Abs to bind specifically torecombinant class I peptide-MHC complexes, as well as to complexespresented on the surface of peptide pulsed APCs.

An important feature of the TCR-like Fab Abs isolated in this study istheir capability to detect TCR ligands at cell surface densities closeto the threshold limit for T cell recognition. The H9HLA-A2/pp65-specific TCR-like Fab Ab was able to detect in areproducible manner as low as 100 sites/cell. Using flow cytometry, itwas possible to use the H9 Fab Ab to detect the specific ligand on cellspulsed with peptide concentrations similar to those required to activateT cell hybridoma or CTL lines to cytokine secretion and within a fewfold of the minimal concentration able to sensitize target cells forlysis in a short-term assay (Porgador A., et al., 1997).

These data indicate that when applied to dissociated cell populationsusing flow cytometry, the detection of ligand with H9 and other TCR-likeFabs with similar affinity approaches the sensitivity of T cells, andhence that these molecules are suitable reagents for evaluatingantigenic complex expression at low, but physiologically relevantlevels. In this study, the detection sensitivity of specific ligand wasobserved with as low as 100 complexes per cell. Thus, this principle hasbeen applied in this study to mixtures of peptide pulsed HLA-A2+ JYcells, and the HLA-A2-B cell line APD. By using the H9 tetramer in asingle-step staining for flow cytometry, it was possible to readilyidentify pp65 495-503 peptide pulsed JY cells admixed with APD cells inas low proportion as 5%.

The avidity of the TCR-like Ab molecules was improved by making therecombinant monovalent molecules into multivalent molecules. This wasfeasible by altering the basic Fab form to a tetrameric molecule or to awhole bivalent IgG Ab.

Detection of class I MHC complexes in association with the pp65 495-503peptide on virus infected cells, showed the ability of the H9 Ab torecognize complexes not only on the surface of peptide pulsed APCs, butalso complexes which were produced by naturally occurring active antigenprocessing. Cytotoxicity assays directed to virus infected cellsconfirmed these findings. The blockage of killing by the CTLs afterincubation with the H9 Ab showed a competition between the cytotoxicT-cell receptor and the H9 TCR-like Ab on the same site presented on thevirus infected cell.

Using the H9 Ab at various time points following infection the presentinventors could track the presentation level of HLA-A2/pp65 complexesduring the course of virus infection cycle. Specific staining with theH9 Ab lead to the observation that the expression level of the specificHLA-A2/pp65 complexes on the cell surface does not represent the overallquantity of these specific complexes, because as shown most of them arelocated inside the cell. The results presented herein demonstrate theexistence of a significant large pool of specific HLA-A2/pp65 complexesinside virus infected cells, which increased as a function of time afterviral infection. The use of a CMV mutant strain which lacks the genesresponsible for MHC class I down regulation revealed similar findings.Large pools of specific complexes, bearing the pp65 495-503 peptide,were found inside the cells. In contrast to the uninfected cells, thereis a large amount of MHC class I complexes inside the cells which areinfected with the wild-type/mutant strain.

The results of the kinetic assays also clearly show that there is agreat correlation between the pp65 expression level and its presentationlevel. Both increase as time goes by. Moreover, the timing of the pp65expression might precede the processing and presentation of thisprotein, as presented in the results.

This work provides also quantitative data about the number of specificHLA-A2/pp65 complexes generated inside infected cells as well aspresented on the cell surface after active intracellular processing byvirus infected cells. The results revealed for the first time the numberof sites which are presented on the cell surface and recognized by theimmune system. Moreover, quantization of general HLA-A2 complexesenabled the present inventors to determine the percentage of complexesdown regulated after viral infection. It also enabled the presentinventors to compare between the number of general complexes and thenumber of specific HLA-A2/pp65 complexes inside the cells and on theirsurface. This analysis enables the determination of the percentage ofthe specific complexes among the general complexes. The resultsindicated quantitatively that most of the complexes inside the virusinfected cells are bearing the pp65 495-503 peptide. Large numbers ofspecific complexes were also found in the cells infected with the mutantstrain, strengthening the previous data, regarding the pools which areprevented from being translocated to the membrane.

Confocal immunofluorescence microscopy enabled for the first time directvisualization of the intracellular and extracellular sites ofpeptide-MHC molecules throughout virus infection cycle, as well asdetermination of their localization inside the cell. This visualizationrevealed the colocalization of the HLA-A2/pp65 complexes with thecis-golgi apparatus. It also showed the exact movement of the complexesfrom this location to the cell surface, in correlation to the virusinfection kinetics. At the progressed time scales there was asignificant display of the complexes on the cell surface.

The study presented here shows the usage of an isolated humanrecombinant Ab towards a specific viral peptide-MHC class I in thefollowing: (i) tracking the level of specific complexes throughout timescale which represents a viral infection cycle; (ii) tracking the numberof complexes throughout time scale inside the cell and on its surfaceand analysis of this data; (iii) visualization of complexes in a viralinfection system which demonstrate the intracellular localization of thecomplexes throughout time scale, and; (iv) detection of the correlationbetween protein expression and its derived peptide presentation onHLA-A2 complexes after processing.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES Additional References are Cited in Text

-   1. Ahn, K. et al. Human cytomegalovirus inhibits antigen    presentation by a sequential multistep process Proc. Natl. Acad.    Sci. U.S.A 93, 10990-10995 (1996).-   2. Allart S, et al., 2003; Invest Ophthalmol V is Sci. 44: 665-71-   3. Altman, J. D. et al. Phenotypic analysis of antigen-specific T    lymphocytes. Science 274, 94-96 (1996).-   4. Chee, M. S. et al. Analysis of the protein-coding content of the    sequence of human cytomegalovirus strain AD169. Cum Top. Microbiol.    Immunol. 154, 125-169 (1990).-   5. Cohen, C. J. et al. Direct detection and quantitation of a    distinct T-cell epitope derived from tumor-specific epithelial    cell-associated mucin using human recombinant antibodies endowed    with the antigen-specific, major histocompatibility    complex-restricted specificity of T cells Cancer Res. 62, 5835-5844    (2002).-   6. Cloutier, S. M. et al. Streptabody, a high avidity molecule made    by tetramerization of in vivo biotinylated, phage display-selected    scFv fragments on streptavidin. Mol. Immunol. 37, 1067-1077 (2000).-   7. De Haard, H. J. et al. A large non-immunized human Fab fragment    phage library that permits rapid isolation and kinetic analysis of    high affinity antibodies. J. Biol. Chem. 274, 18218-18230 (1999).-   8. Denkberg, G., Cohen, C. J., Segal, D., Kirkin, A. F. & Reiter, Y.    Recombinant human single-chain MHC-peptide complexes made from E.    coli By in vitro refolding: functional single-chain MHC-peptide    complexes and tetramers with tumor associated antigens. Eur. J.    Immunol. 30, 3522-3532 (2000).-   9. Denkberg, G., Cohen, C. J. & Reiter, Y. Critical role for CD8 in    binding of MHC tetramers to TCR: CD8 antibodies block specific    binding of human tumor-specific MHC-peptide tetramers to TCR. J.    Immunol. 167, 270-276 (2001).-   10. Denkberg, G. et al. Direct visualization of distinct T cell    epitopes derived from a melanoma tumor-associated antigen by using    human recombinant antibodies with MHC-restricted T cell    receptor-like specificity. Proc. Natl. Acad. Sci. U.S.A 99,    9421-9426 (2002).-   11. Jones, T. R. & Sun, L. Human cytomegalovirus US2 destabilizes    major histocompatibility complex class I heavy chains J. Virol. 71,    2970-2979 (1997).-   12. Lee, P. P. et al. Characterization of circulating T cells    specific for tumor-associated antigens in melanoma patients. Nat Med    5, 677-685 (1999).-   13. Lev, A. et al. Isolation and characterization of human    recombinant antibodies endowed with the antigen-specific, major    histocompatibility complex-restricted specificity of T cells    directed toward the widely expressed tumor T-cell epitopes of the    telomerase catalytic subunit. Cancer Res. 62, 3184-3194 (2002).-   14. Pascolo, S. et al. HLA-A2.1-restricted education and cytolytic    activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m)    HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice. J.    Exp. Med. 185, 2043-2051 (1997).-   15. Porgador, A., Yewdell, J. W., Deng, Y. P., Bennink, J. R. &    Germain, R.N. Localization, quantitation, and in situ detection of    specific peptide MHC class I complexes using a monoclonal antibody.    Immunity 6, 715-726 (1997).-   16. Soderberg-Naucler, C., Fish, K. N. & Nelson, J. A. Growth of    human cytomegalovirus in primary macrophages. Methods 16,    126-+(1998).

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130101594A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. An antibody comprising an antigen recognitiondomain capable of binding an MHC molecule being complexed with acytomegalovirus (CMV) pp65 or pp64 peptide as set forth by SEQ ID NO:3,wherein the antibody does not bind said MHC molecule in an absence ofsaid complexed peptide, and wherein the antibody does not bind saidpeptide in an absence of said MHC molecule.
 2. The antibody of claim 1,being conjugated to a therapeutic moiety.
 3. The antibody of claim 1,attached to a detectable moiety.
 4. The antibody of claim 1, being anantibody fragment.
 5. An antibody comprising a multivalent form of theantibody of claim
 1. 6. The antibody of claim 5, wherein saidmultivalent form is an IgG antibody.
 7. A pharmaceutical compositioncomprising as an active ingredient the antibody of claim
 1. 8. A methodof detecting a cell expressing a cytomegalovirus (CMV) antigen,comprising contacting the cell with the antibody of claim 1 underconditions which allow immunocomplex formation, wherein a presence or alevel above a predetermined threshold of said immunocomplex isindicative of CMV expression in the cell.
 9. A method of diagnosing acytomegalovirus (CMV) infection in a subject in need thereof, comprisingcontacting a cell of the subject with the antibody of claim 1 underconditions which allow immunocomplex formation, wherein a presence or alevel above a pre-determined threshold of said immunocomplex in the cellis indicative of the CMV infection in the subject.
 10. A method oftreating a disease associated with cytomegalovirus (CMV) infection,comprising administering to a subject in need thereof a therapeuticallyeffective amount of the antibody of claim 1, thereby treating thedisease associated with CMV infection.
 11. The method of claim 9,wherein said subject has a suppressed or a compromised immune system.12. The method of claim 9, wherein said CMV infection is associated witha disease selected from the group consisting of mononucleosis,retinitis, pneumonia, gastrointestinal disorders, and encephalitis. 13.The method of claim 9, wherein said cell is a retina cell, lungepithelial cell, a gastrointestinal epithelial cell or a brain cell. 14.The method of claim 9, wherein said subject is an immuno-compromisedorgan transplant recipient.
 15. The method of claim 9, wherein saidsubject is infected with human immunodeficiency virus (HIV).